CO2 starvation experiments provide support for the carbon-limited hypothesis on the evolution of CAM-like behaviour in Isoëtes

Background and AimsCrassulacean acid metabolism (CAM) is an adaptation to increase water use efficiency in dry environments. Similar biochemical patterns occur in the aquatic lycophyte genus Isoëtes. It has long been assumed and accepted that CAM-like behaviour in these aquatic plants is an adaptation to low daytime carbon levels in aquatic ecosystems, but this has never been directly tested.MethodsTo test this hypothesis, populations of Isoëtes engelmannii and I. tuckermanii were grown in climate-controlled chambers and starved of atmospheric CO₂ during the day while pH was measured for 24 h.Key ResultsWe demonstrate that terrestrial plants exposed to low atmospheric CO₂ display diel acidity cycles similar to those in both xerophytic CAM plants and submerged Isoëtes.ConclusionsDaytime CO₂ starvation induces CAM-like nocturnal acid accumulation in terrestrial Isoëtes, substantiating the hypothesis that carbon starvation is a selective pressure for this physiological behaviour.     

Trading Off Global Fuel Supply,CO2 Emissions and Sustainable Development

The United Nations Conference on Climate Change (Paris 2015) reached an international agreement to keep the rise in global average temperature ‘well below 2°C’ and to ‘aim to limit the increase to 1.5°C’. These reductions will have to be made in the face of rising global energy demand. Here a thoroughly validated dynamic econometric model (Eq 1) is used to forecast global energy demand growth (International Energy Agency and BP), which is driven by an increase of the global population (UN), energy use per person and real GDP (World Bank and Maddison). Even relatively conservative assumptions put a severe upward pressure on forecast global energy demand and highlight three areas of concern. First, is the potential for an exponential increase of fossil fuel consumption, if renewable energy systems are not rapidly scaled up. Second, implementation of internationally mandated CO₂ emission controls are forecast to place serious constraints on fossil fuel use from ~2030 onward, raising energy security implications. Third is the challenge of maintaining the international ‘pro-growth’ strategy being used to meet poverty alleviation targets, while reducing CO₂ emissions. Our findings place global economists and environmentalists on the same side as they indicate that the scale up of CO₂ neutral renewable energy systems is not only important to protect against climate change, but to enhance global energy security by reducing our dependence of fossil fuels and to provide a sustainable basis for economic development and poverty alleviation. Very hard choices will have to be made to achieve ‘sustainable development’ goals.     

Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

     

Respiratory CO(2) as Carbon Source for Nocturnal Acid Synthesis at High Temperatures in Three Species Exhibiting Crassulacean Acid Metabolism

Temperature effects on nocturnal carbon gain and nocturnal acid accumulation were studied in three species of plants exhibiting Crassulacean acid metabolism: Mamillaria woodsii, Opuntia vulgaris, and Kalanchoë daigremontiana. Under conditions of high soil moisture, nocturnal CO₂ gain and acid accumulation had temperature optima at 15 to 20°C. Between 5 and 15°C, uptake of atmospheric CO₂ largely accounted for acid accumulation. At higher tissue temperatures, acid accumulation exceeded net carbon gain indicating that acid synthesis was partly due to recycling of respiratory CO₂. When plants were kept in CO₂-free air, acid accumulation based on respiratory CO₂ was highest at 25 to 35°C. Net acid synthesis occurred up to 45°C, although the nocturnal carbon balance became largely negative above 25 to 35°C. Under conditions of water stress, net CO₂ exchange and nocturnal acid accumulation were reduced. Acid accumulation was proportionally more decreased at low than at high temperatures. Acid accumulation was either similar over the whole temperature range (5-45°C) or showed an optimum at high temperatures, although net carbon balance became very negative with increasing tissue temperatures. Conservation of carbon by recycling respiratory CO₂ was temperature dependent. At 30°C, about 80% of the dark respiratory CO₂ was conserved by dark CO₂ fixation, in both well irrigated and water stressed plants.     

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.Subject terms: Environmental microbiology, Metabolism     

Diel shifts in carboxylation pathway and metabolite dynamics in the CAM bromeliad Aechmea 'Maya' in response to elevated CO2

Background and Aims

The deployment of temporally separated carboxylation pathways for net CO₂ uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO₂ world. The present study examined how long-term exposure to elevated CO₂ influences the relative contributions that C₃ and C₄ carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle.

Methods

Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea ‘Maya’ after exposure to 700 µmol mol⁻¹ CO₂ for 5 months.

Key Results

There was a 60 % increase in 24-h carbon gain under elevated CO₂ due to a stimulation of daytime C₃ and C₄ carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO₂ taken up under elevated CO₂ was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO₂ there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate.

Conclusions

Despite the increased size of the soluble sugar storage pool under elevated CO₂, there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. ‘Maya’. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO₂ in this CAM bromeliad.Key words: Aechmea ‘Maya’, carbon budgets, elevated CO₂, gas exchange, metabolite dynamics, PEPC, photosynthetic plasticity, Rubisco     

The pathway of carbon dioxide fixation in crassulacean plants

Combined gas chromatography-mass spectrometry of malic acid derivatives has been used to show unequivocally that malic acid, synthesized during active acid accumulation in the dark by Kalanchoë daigremontiana Hammet et Perrier in the presence of ¹³CO₂ is produced by a pathway involving a single carboxylation. The significance of the finding that crassulacean malate synthesized in the dark and in the presence of ¹⁴CO₂ often contains 66% of the total carboxyl label in carbon atom 4, which has previously been taken to indicate the operation of a double carboxylation pathway or has been dismissed as an artefact, is discussed.     

Differential Responses of CO2 Assimilation,Carbohydrate Allocation and Gene Expression to NaCl Stress in Perennial Ryegrass with Different Salt Tolerance

Little is known about the effects of NaCl stress on perennial ryegrass (Lolium perenne L.) photosynthesis and carbohydrate flux. The objective of this study was to understand the carbohydrate metabolism and identify the gene expression affected by salinity stress. Seventy-four days old seedlings of two perennial ryegrass accessions (salt-sensitive ‘PI 538976’ and salt-tolerant ‘Overdrive’) were subjected to three levels of salinity stress for 5 days. Turf quality in all tissues (leaves, stems and roots) of both grass accessions negatively and significantly correlated with GFS (Glu+Fru+Suc) content, except for ‘Overdrive’ stems. Relative growth rate (RGR) in leaves negatively and significantly correlated with GFS content in ‘Overdrive’ (P<0.01) and ‘PI 538976’ (P<0.05) under salt stress. ‘Overdrive’ had higher CO₂ assimilation and F_v/F_m than ‘PI 538976’. Intercellular CO₂ concentration, however, was higher in ‘PI 538976’ treated with 400 mM NaCl relative to that with 200 mM NaCl. GFS content negatively and significantly correlated with RGR in ‘Overdrive’ and ‘PI 538976’ leaves and in ‘PI 538976’ stems and roots under salt stress. In leaves, carbohydrate allocation negatively and significantly correlated with RGR (r² = 0.83, P<0.01) and turf quality (r² = 0.88, P<0.01) in salt-tolerant ‘Overdrive’, however, the opposite trend for salt-sensitive ‘PI 538976’ (r² = 0.71, P<0.05 for RGR; r² = 0.62, P>0.05 for turf quality). A greater up-regulation in the expression of SPS, SS, SI, 6-SFT gene was observed in ‘Overdrive’ than ‘PI 538976’. A higher level of SPS and SS expression in leaves was found in ‘PI 538976’ relative to ‘Overdrive’. Accumulation of hexoses in roots, stems and leaves can induce a feedback repression to photosynthesis in salt-stressed perennial ryegrass and the salt tolerance may be changed with the carbohydrate allocation in leaves and stems.     

Short- and long-term conditioning of a temperate marine diatom community to acidification and warming

Ocean acidification and greenhouse warming will interactively influence competitive success of key phytoplankton groups such as diatoms, but how long-term responses to global change will affect community structure is unknown. We incubated a mixed natural diatom community from coastal New Zealand waters in a short-term (two-week) incubation experiment using a factorial matrix of warming and/or elevated pCO₂ and measured effects on community structure. We then isolated the dominant diatoms in clonal cultures and conditioned them for 1 year under the same temperature and pCO₂ conditions from which they were isolated, in order to allow for extended selection or acclimation by these abiotic environmental change factors in the absence of interspecific interactions. These conditioned isolates were then recombined into ‘artificial’ communities modelled after the original natural assemblage and allowed to compete under conditions identical to those in the short-term natural community experiment. In general, the resulting structure of both the unconditioned natural community and conditioned ‘artificial’ community experiments was similar, despite differences such as the loss of two species in the latter. pCO₂ and temperature had both individual and interactive effects on community structure, but temperature was more influential, as warming significantly reduced species richness. In this case, our short-term manipulative experiment with a mixed natural assemblage spanning weeks served as a reasonable proxy to predict the effects of global change forcing on diatom community structure after the component species were conditioned in isolation over an extended timescale. Future studies will be required to assess whether or not this is also the case for other types of algal communities from other marine regimes.     

An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions.     

Complex Spatiotemporal Responses of Global Terrestrial Primary Production to Climate Change and Increasing Atmospheric CO2 in the 21st Century

Quantitative information on the response of global terrestrial net primary production (NPP) to climate change and increasing atmospheric CO₂ is essential for climate change adaptation and mitigation in the 21^st century. Using a process-based ecosystem model (the Dynamic Land Ecosystem Model, DLEM), we quantified the magnitude and spatiotemporal variations of contemporary (2000s) global NPP, and projected its potential responses to climate and CO₂ changes in the 21^st century under the Special Report on Emission Scenarios (SRES) A2 and B1 of Intergovernmental Panel on Climate Change (IPCC). We estimated a global terrestrial NPP of 54.6 (52.8–56.4) PgC yr⁻¹ as a result of multiple factors during 2000–2009. Climate change would either reduce global NPP (4.6%) under the A2 scenario or slightly enhance NPP (2.2%) under the B1 scenario during 2010–2099. In response to climate change, global NPP would first increase until surface air temperature increases by 1.5°C (until the 2030s) and then level-off or decline after it increases by more than 1.5°C (after the 2030s). This result supports the Copenhagen Accord Acknowledgement, which states that staying below 2°C may not be sufficient and the need to potentially aim for staying below 1.5°C. The CO₂ fertilization effect would result in a 12%–13.9% increase in global NPP during the 21^st century. The relative CO₂ fertilization effect, i.e. change in NPP on per CO₂ (ppm) bases, is projected to first increase quickly then level off in the 2070s and even decline by the end of the 2080s, possibly due to CO₂ saturation and nutrient limitation. Terrestrial NPP responses to climate change and elevated atmospheric CO₂ largely varied among biomes, with the largest increases in the tundra and boreal needleleaf deciduous forest. Compared to the low emission scenario (B1), the high emission scenario (A2) would lead to larger spatiotemporal variations in NPP, and more dramatic and counteracting impacts from climate and increasing atmospheric CO₂.     

Effect of Varying CO(2) Partial Pressure on Photosynthesis and on Carbon Isotope Composition of Carbon-4 of Malate from the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana Hamet et Perr

Intact leaves of Kalanchoë daigremontiana were exposed to CO₂ partial pressures of 100, 300, and 1000 microbars. Malic acid was extracted, purified, and degraded in order to obtain isotopic composition of carbon-1 and carbon-4. From these data, it is possible to calculate the carbon isotope composition of newly fixed carbon in malate. In all three treatments, the isotopic composition of newly introduced carbon is the same as that of the CO₂ source and is independent of CO₂ partial pressures over the range tested. Comparison with numerical models described previously (O'Leary 1981 Phytochemistry 20: 553-567) indicates that we would expect carbon 4 of malate to be 4‰ more negative than source CO₂ if diffusion is totally limiting or 7‰ more positive than source CO₂ if carboxylation is totally limiting. Our results demonstrate that stomatal aperture adjusts to changing CO₂ partial pressures and maintains the ratio of diffusion resistance to carboxylation resistance approximately constant. In this study, carboxylation and diffusion resistances balance so that essentially no fractionation occurs during malate synthesis. Gas exchange studies of the same leaves from which malate was extracted show that the extent of malate synthesis over the whole night is nearly independent of CO₂ partial pressure, although there are small variations in CO₂ uptake rate. Both the gas exchange and the isotope studies indicate that the ratio of external to internal CO₂ partial pressure is the same in all three treatments. Inasmuch as a constant ratio will result in constant isotope fractionation, this observation may explain why plants in general have fairly invariable ¹³C contents, despite growing under a variety of environmental conditions.     

Giemsa-Stained Wet Mount Based Method for Reticulocyte Quantification: A Viable Alternative in Resource Limited or Malaria Endemic Settings

The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on ‘subvital’ staining. The specialist ‘subvital’ stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa''s stain (commonly used microbiology and parasitology) in a ‘subvital’ manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.     

Modulation of Rubisco Activity during the Diurnal Phases of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

The regulation of Rubisco activity was investigated under high, constant photosynthetic photon flux density during the diurnal phases of Crassulacean acid metabolism in Kalanchoë daigremontiana Hamet et Perr. During phase I, a significant period of nocturnal, C₄-mediated CO₂ fixation was observed, with the generated malic acid being decarboxylated the following day (phase III). Two periods of daytime atmospheric CO₂ fixation occurred at the beginning (phase II, C₄–C₃ carboxylation) and end (phase IV, C₃–C₄ carboxylation) of the day. During the 1st h of the photoperiod, when phosphoenolpyruvate carboxylase was still active, the highest rates of atmospheric CO₂ uptake were observed, coincident with the lowest rates of electron transport and minimal Rubisco activity. Over the next 1 to 2 h of phase II, carbamylation increased rapidly during an initial period of decarboxylation. Maximal carbamylation (70%–80%) was reached 2 h into phase III and was maintained under conditions of elevated CO₂ resulting from malic acid decarboxylation. Initial and total Rubisco activity increased throughout phase III, with maximal activity achieved 9 h into the photoperiod at the beginning of phase IV, as atmospheric CO₂ uptake recommenced. We suggest that the increased enzyme activity supports assimilation under CO₂-limited conditions at the start of phase IV. The data indicate that Rubisco activity is modulated in-line with intracellular CO₂ supply during the daytime phases of Crassulacean acid metabolism.     

Mechanisms of Attenuation of Pulmonary V’O2 Slow Component in Humans after Prolonged Endurance Training

In this study we have examined the effect of prolonged endurance training program on the pulmonary oxygen uptake (V’O₂) kinetics during heavy-intensity cycling-exercise and its impact on maximal cycling and running performance. Twelve healthy, physically active men (mean±SD: age 22.33±1.44 years, V’O_2peak 3198±458 mL ∙ min^-1) performed an endurance training composed mainly of moderate-intensity cycling, lasting 20 weeks. Training resulted in a decrease (by ~5%, P = 0.027) in V’O₂ during prior low-intensity exercise (20 W) and in shortening of τ_p of the V’O₂ on-kinetics (30.1±5.9 s vs. 25.4±1.5 s, P = 0.007) during subsequent heavy-intensity cycling. This was accompanied by a decrease of the slow component of V’O₂ on-kinetics by 49% (P = 0.001) and a decrease in the end-exercise V’O₂ by ~5% (P = 0.005). An increase (P = 0.02) in the vascular endothelial growth factor receptor 2 mRNA level and a tendency (P = 0.06) to higher capillary-to-fiber ratio in the vastus lateralis muscle were found after training (n = 11). No significant effect of training on the V’O_2peak was found (P = 0.12). However, the power output reached at the lactate threshold increased by 19% (P = 0.01). The power output obtained at the V’O_2peak increased by 14% (P = 0.003) and the time of 1,500-m performance decreased by 5% (P = 0.001). Computer modeling of the skeletal muscle bioenergetic system suggests that the training-induced decrease in the slow component of V’O₂ on-kinetics found in the present study is mainly caused by two factors: an intensification of the each-step activation (ESA) of oxidative phosphorylation (OXPHOS) complexes after training and decrease in the ‘‘additional” ATP usage rising gradually during heavy-intensity exercise.     

Carbon Dioxide and Water Vapor Exchange in the Crassulacean Acid Metabolism Plant Kalanchoë pinnáta during a Prolonged Light Period: METABOLIC AND STOMATAL CONTROL OF CARBON METABOLISM

Net CO₂ and water vapor exchange were studied in the Crassulacean acid metabolism plant Kalanchoë pinnáta during a normal 12-hour light/12-hour dark cycle and during a prolonged light period. Leaf temperature and leaf-air vapor pressure difference were kept constant at 20 C and 9 to 10 millibar. There was a 25% increase in the rate of CO₂ fixation during the first 6 hours prolonged light without change in stomatal conductance. This was associated with a decrease in the intracellular partial pressure of CO₂, a decrease in the stimulation of net CO₂ uptake by 2% O₂, and a decrease in the CO₂ compensation point from 45 to 0 microbar. In the normal light period after deacidification, leaves showed a normal light dependence of CO₂ uptake but, in prolonged light, CO₂ uptake was scarcely light-dependent. The increase in titratable acidity in prolonged light was similar to that in the dark.     

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO(2) Fixation in Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPc) catalyzes the primary fixation of CO₂ in Crassulacean acid metabolism plants. Flux through the enzyme is regulated by reversible phosphorylation. PEPc kinase is controlled by changes in the level of its translatable mRNA in response to a circadian rhythm. The physiological significance of changes in the levels of PEPc-kinase-translatable mRNA and the involvement of metabolites in control of the kinase was investigated by subjecting Kalanchoë daigremontiana leaves to anaerobic conditions at night to modulate the magnitude of malate accumulation, or to a rise in temperature at night to increase the efflux of malate from vacuole to cytosol. Changes in CO₂ fixation and PEPc kinase activity reflected those in kinase mRNA. The highest rates of CO₂ fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. In leaves subjected to anaerobic treatment overnight and transferred to ambient air at the start of the day, PEPc-kinase-translatable mRNA and activity, the phosphorylation state of PEPc, and fixation of atmospheric CO₂ were significantly higher than those for control leaves for the first 3 h of the light period. A nighttime temperature increase from 19°C to 27°C led to a rapid reduction in kinase mRNA and activity; however, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These data are consistent with the hypothesis that a high concentration of malate reduces both kinase mRNA and the accumulation of the kinase itself.     

Introgression and confirmation of everbearing trait in strawberry (Fragaria x ananassa Duch.)

The present investigation was targeted towards a highly desirable everbearing trait in strawberry (Fragaria × ananassa Duch.) via marker assisted selection while seeing its worldwide commercial applicability through the extended harvest season. The crosses were made between everbearing and june-bearing cultivars to raise the F₁ individuals. Morphological characters (plant, floral, and fruit) were assessed that showed significant differences among the strawberry cultivars. Molecular characterization was carried out between everbearing and non-everbearing cultivars using RAPD and SSR markers. For phenotyping, a chi-square test was performed and revealed that out of all four cross combinations, the best fitted cross found to be in Mendelian segregation ratio (1:1) was ‘Confectura’ × ‘Torrey’ with χ²-value 1.58. Further, the identified polymorphic markers were assessed across the F₁ individuals of cross ‘Confectura’ × ‘Torrey’ for its genotyping. It could be revealed that the targeted everbearing trait is governed by a dominant gene(s) in the subjected strawberry genotypes. Further, the identified polymorphic markers would be successfully employed in DNA fingerprinting of strawberry under various crop improvement programme.Supplementary informationThe online version of this article (10.1007/s12298-020-00916-w) contains supplementary material, which is available to authorized users.     

Optimization of linkage mapping strategy and construction of a high-density American lotus linkage map

Background

Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification.

Results

We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F₁ population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome.

Conclusions

Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.