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Transcriptomic changes in plants during drought stress give insights into the mechanisms with which plants stabilise their metabolic processes in order to cope with the drought condition. In potato, understanding such drought-induced transcriptomic changes is critical because prolonged field drought interferes with tuber formation and bulking period of potato development, which eventually affects yield. We hypothesised that phenotypic drought responses of potato genotypes may be linked to differences in transcriptomic changes. Using an RNA sequencing approach, we investigated such transcriptomic changes in leaves of three cultivars (Biogold, Hansa and Lady Rosetta) under drought. We found more differentially expressed genes (DEGs) in the tolerant cultivars, Lady Rosetta and Biogold, than in the sensitive cultivar (Hansa). The differential gene expression trend reflected the phenotypic drought responses of the cultivars. For instance, we found in both Biogold and Lady Rosetta but not in Hansa, an upregulation of genes involved in carbohydrate metabolism (e.g., Alpha-glucosidase), flavonoid biosynthesis (e.g., Flavanone 3 beta-hydroxylase), lipid biosynthesis/transfer (e.g., nonspecific Lipid Transfer Proteins), heat shock proteins and secondary metabolites like phenolics and lignins. Furthermore, a prolonged drought stress resulted in reduced DEGs in Biogold and Hansa, but not in Lady Rosetta that also maintained its tuber yield under such prolonged stress suggesting a more robust drought tolerance. Our findings suggest that a synergistic expression of genes involved in several different aspects of drought response is required in order to obtain a robust tolerance.

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot- or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further functional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+-induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.

Plants have evolved to deal with different stresses during plant growth, relying on complex interactions or crosstalk between multiple signalling pathways in plant cells. In this sophisticated regulatory network, Ca2+ transients in the cytosol ([Ca2+]cyt) act as major physiological signals to initiate appropriate responses. The CALCINEURIN B-LIKE PROTEIN (CBL)-CBL-INTERACTING PROTEIN KINASE (CIPK) network relays physiological signals characterised by [Ca2+]cyt transients during plant development and in response to environmental changes. Many studies are aimed at elucidating the role of the CBL-CIPK network in plant growth and stress responses. This review discusses the involvement of the CBL-CIPK pathways in two levels of crosstalk between plant development and stress adaptation: direct crosstalk through interaction with regulatory proteins, and indirect crosstalk through adaptation of correlated physiological processes that affect both plant development and stress responses. This review thus provides novel insights into the physiological roles of the CBL-CIPK network in plant growth and stress adaptation.

Deciphering the genetic basis of complex traits like nitrogen use efficiency (NUE) requires understanding the genotype-by-environment (G × E) interaction and linking physiological functions and agronomic traits to DNA markers. Multi-environment experiments were conducted in different environments representing low and high nitrogen levels combined with rain-fed and irrigation production conditions at three different locations in Ethiopia: Debre-Tabor, Injibara and Koga, in 2013 and 2015. The objectives of the study were to determine the G × E interaction and stability of genotypes for NUE of potato and to identify markers associated with NUE and NUE-related agronomic and physiological traits in potato under these diverse environments. Data were analyzed using GenStat, and genotype plus the genotype and environment (GGE) biplot model; the marker-trait associations were discovered using the R-software package GWASpoly. The analysis of variance that included location and production system had estimates for genotype variance (σ2g) that were low compared with the estimates for environment variance (σ2e) and genotype-by-environment interaction variance (σ2ge) for most measured traits at both N levels. The GGE analysis identified two mega-environments that coincided with the two production systems. The high N level environments both at Debre-Tabor and Injibara, and the low N environment at Koga, respectively, were the most suitable environments for discriminating the potato cultivars and being representative test environments for NUE evaluation in the rain-fed mega-environment and irrigation mega-environment. A total of 77 marker trait associations were identified for NUE and NUE-related agronomic and physiological traits. Multi-trait genomic regions that harbored significant marker-trait associations for NUE and NUE-related traits were found on chromosomes III, V and VI. The effect of production season was greater than the effect of N levels on QTL × environment interaction for most NUE-related traits.

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

Developing N use efficient potato varieties requires exploring the genetic basis of nitrogen use efficiency (NUE) and associated agronomic and physiological traits. In order to identify QTLs for NUE and NUE-related traits, and to determine the relationships between the traits and QTLs in potato, a diploid potato mapping population (CxE) was evaluated in the field in Ethiopia under low and high N fertilizer levels. QTL detection was performed using interval mapping and multiple QTL mapping (MQM). A total of 52 putative QTLs were identified for ten traits, of which 28 QTLs were detected under low N availability while the remaining 24 QTLs were detected under high N conditions. Several QTLs were location and N level specific, suggesting the presence of QTL x environment interaction. A region on linkage group V (21-38 cM) accumulated the largest number of QTLs. This region coincides with the earliness locus encoded by the CDF1 gene, suggesting that earliness has a profound influence on NUE. A putative second QTL region on linkage group V located 20 cM from the earliness locus (38-56 cM) and a region on linkage group IV (60-72 cM) might be useful other regions to focus on, for NUE improvement in potato. To verify the stability of the identified QTLs and to use these for the detection of possible candidate genes, further multi-environment trials with larger population size may be required.

Potato (Solanum tuberosum) is an important food crop consumed all over the world, but it is generally sensitive to drought conditions. One of the major physiological processes affected by drought stress is carbon partitioning: the plant's choice of where to allocate its photoassimilates. Our aim was to investigate the molecular factors and possible bottlenecks affecting carbon partitioning during drought. We studied potato cultivars with contrasting drought responses in the greenhouse in the years 2013–2015, and further investigated the expression of genes involved in carbon partitioning and metabolite levels. Our results indicate that one of the most severe effects of drought stress on potato is the arrest of stolon differentiation and formation of tubers. We also identified some physiological traits like stomatal conductance and chlorophyll content as affecting carbon assimilation, partitioning and eventual tuber yield. The gene expressions and biochemical analyses highlight the various tissues prioritized by the plant for assimilate transport during drought stress, and give indications of what distinguishes drought tolerance and sensitivity of cultivated potato. Some of the key genes studied (like Sucrose synthase and Sucrose transporters) may be inclusive breeding targets for drought tolerance in potato.

Developing cultivars that use nitrogen more efficiently is a sustainable strategy for reducing nitrogen use in crop production. To assess the genetic diversity for nitrogen use efficiency (NUE) and related traits in potato, a total of 97 (88 for the Western-European market and 9 Ethiopian) cultivars were evaluated at two nitrogen levels (40 kg/ha and 120 kg/ha) for 24 quantitative traits in Debre-Tabor and Injibara (Ethiopia) in the 2013 main rainy season (June to September). Highly significant variation was found among genotypes for almost all measured traits. Plant height, NUE, tuber yield, and yield-related traits and model parameters for canopy development (maximum canopy covers area under the canopy curve) were significantly affected by N levels across locations. Dutch cultivars had more rapid initial canopy development and matured earlier than the Ethiopian cultivars at both N levels and locations. A hierarchical cluster analysis grouped the cultivars in 9 and 11 genetically distinct classes at low and high N, respectively. The genetic component accounted for a large portion of the phenotypic variation for plant height, tuber number per plant, average tuber weight, and NUE under both N regimes, as indicated by a high heritability. Strong phenotypic correlations were observed between NUE and tuber number per plant, days to maturity, tuber dry matter %, maximum canopy cover, and area under the canopy curve under both low and high N conditions. The result is indicative to set the best parental line selection criteria for crossing purpose and utilize the cultivars for further potato NUE breeding programmes.

The Plantarray 3.0 phenotyping platform® was used to monitor the growth and water use of the quinoa varieties Pasto and selRiobamba under salinity (0–300 mM NaCl). Salinity reduced the cumulative transpiration of both varieties by 60% at 200 mM NaCl and by 75 and 82% at 300 mM NaCl for selRiobamba and Pasto, respectively. Stomatal conductance was reduced by salinity, but at 200 mM NaCl Pasto showed a lower reduction (15%) than selRiobamba (35%), along with decreased specific leaf area. Diurnal changes in water use parameters indicate that under salt stress, daily transpiration in quinoa is less responsive to changes in light irradiance, and stomatal conductance is modulated to maximize CO2 uptake and minimize water loss following the changes in VPD (vapor pressure deficit). These changes might contribute to the enhanced water use efficiency of both varieties under salt stress. The mechanistic crop model LINTUL was used to integrate physiological responses into the radiation use efficiency of the plants (RUE), which was more reduced in Pasto than selRiobamba under salinity. By the end of the experiment (eleven weeks after sowing, six weeks after stress), the growth of Pasto was significantly lower than selRiobamba, fresh biomass was 50 and 35% reduced at 200 mM and 70 and 50% reduced at 300 mM NaCl for Pasto and selRiobamba, respectively. We argue that contrasting water management strategies can at least partly explain the differences in salt tolerance between Pasto and selRiobamba. Pasto adopted a “conservative-growth” strategy, saving water at the expense of growth, while selRiobamba used an “acquisitive-growth” strategy, maximizing growth in spite of the stress. The implementation of high-resolution phenotyping could help to dissect these complex growth traits that might be novel breeding targets for abiotic stress tolerance.

Many tobacco (Nicotiana tabacum) cultivars are salt-tolerant and thus are potential model plants to study the mechanisms of salt stress tolerance. The CALCINEURIN B-LIKE PROTEIN (CBL) is a vital family of plant calcium sensor proteins that can transmit Ca2+ signals triggered by environmental stimuli including salt stress. Therefore, assessing the potential of NtCBL for genetic improvement of salt stress is valuable. In our studies on NtCBL members, constitutive overexpression of NtCBL5A was found to cause salt supersensitivity with necrotic lesions on leaves. NtCBL5A-overexpressing (OE) leaves tended to curl and accumulated high levels of reactive oxygen species (ROS) under salt stress. The supersensitivity of NtCBL5A-OE leaves was specifically induced by Na+, but not by Cl−, osmotic stress, or drought stress. Ion content measurements indicated that NtCBL5A-OE leaves showed sensitivity to the Na+ accumulation levels that wild-type leaves could tolerate. Furthermore, transcriptome profiling showed that many immune response-related genes are significantly upregulated and photosynthetic machinery-related genes are significantly downregulated in salt-stressed NtCBL5A-OE leaves. In addition, the expression of several cation homeostasis-related genes was also affected in salt-stressed NtCBL5A-OE leaves. In conclusion, the constitutive overexpression of NtCBL5A interferes with the normal salt stress response of tobacco plants and leads to Na+-dependent leaf necrosis by enhancing the sensitivity of transgenic leaves to Na+. This Na+ sensitivity of NtCBL5A-OE leaves might result from the abnormal Na+ compartmentalization, plant photosynthesis, and plant immune response triggered by the constitutive overexpression of NtCBL5A. Identifying genes and pathways involved in this unusual salt stress response can provide new insights into the salt stress response of tobacco plants.

Quinoa is a nutritious seed crop with a great potential to grow in saline soils. Here, we studied ion concentrations in quinoa tissues throughout the life cycle of the plant, and linked ion dynamics to responses in growth parameters, seed yield and efficiency of photosynthesis under salinity (0–400 mM NaCl). Ion dynamics changed from high ion exclusion (>99 %, root contents lower than root medium and low accumulation of ions in the leaves) before flowering, to a build-up of ions during seed filling. This indicates a change in strategy in maintaining the necessary gradient of water potential from the root medium to the leaves. K+ concentrations in leaves also increased by more than 100 % in response to prolonged severe salt stress, which may point to a role of this ion in leaf osmotic adjustment. Accumulation of ions in epidermal bladder cells did not contribute substantially to Na+-exclusion as it was less than 6 % of the total Na+ taken up in leaves. Growth under salt stress was mostly impaired by anatomical adaptations (reduced SLA), while initial light use efficiency (Fv/Fm) and NAR were not affected. The variety Pasto showed a “survival strategy” to high salinity with higher ion exclusion and a higher reduction in transpiration than the other varieties, at the expense of lower biomass and seed yield.

Main conclusionAdaptation of the xylem under dehydration to smaller sized vessels and the increase in xylem density per stem area facilitate water transport during water-limiting conditions, and this has implications for assimilate transport during drought.AbstractThe potato stem is the communication and transport channel between the assimilate-exporting source leaves and the terminal sink tissues of the plant. During environmental stress conditions like water scarcity, which adversely affect the performance (canopy growth and tuber yield) of the potato plant, the response of stem tissues is essential, however, still understudied. In this study, we investigated the response of the stem tissues of cultivated potato grown in the greenhouse to dehydration using a multidisciplinary approach including physiological, biochemical, morphological, microscopic, and magnetic resonance imaging techniques. We observed the most significant effects of water limitation in the lower stem regions of plants. The light microscopy analysis of the potato stem sections revealed that plants exposed to this particular dehydration stress have higher total xylem density per unit area than control plants. This increase in the total xylem density was accompanied by an increase in the number of narrow-diameter xylem vessels and a decrease in the number of large-diameter xylem vessels. Our MRI approach revealed a diurnal rhythm of xylem flux between day and night, with a reduction in xylem flux that is linked to dehydration sensitivity. We also observed that sink strength was the main driver of assimilate transport through the stem in our data set. These findings may present potential breeding targets for drought tolerance in potato.

Background: Potato is highly valued as a food security crop but is sensitive to drought stress. A relatively small reduction of the optimum amount of water can already result in a significant reduction in tuber yield. Therefore, unravelling the genetic basis of drought tolerance is important in order to enhance tolerance to drought in potato. We have evaluated a diploid potato backcross mapping population for drought tolerance under field conditions. Water application was withheld at the stage of tuber initiation. At the end of the drought stress period, we collected data on a number of morphological and physiological traits.Results: The potato population showed significant reduction in several growth traits measured, as well as in tuber yield. We applied quantitative trait locus (QTL) analysis to find genomic regions underlying these drought tolerance traits. We found 60 QTL for several of the traits measured under well-watered and drought stress conditions. From the total number of QTLs identified, 21 were detected under water stress conditions while 39 QTLs were identified under well-watered conditions. Drought specific QTLs for tuber fresh yield was detected on chromosome 7, 9 and 12.Conclusions: There is genetic variation in potato for drought tolerance and QTLs discovered for drought tolerance traits in this study can aid in improving potato for drought tolerance.

In plants, tolerance to drought stress is a result of numerous minor effect loci in which transcriptional regulation contributes significantly to the observed phenotypes. Under severe drought conditions, a major expression quantitative trait loci hotspot was identified on chromosome five in potato. A putative Nuclear factor y subunit C4 was identified as key candidate in the regulatory cascade in response to drought. Further investigation of the eQTL hotspots suggests a role for a putative Homeobox leucine zipper protein 12 in relation to drought in potato. Genes strongly co-expressed with Homeobox leucine zipper protein 12 were plant growth regulators responsive to water deficit stress in Arabidopsis thaliana, implying a possible conserved mechanism. Integrative analysis of genetic, genomic, phenotypic and transcriptomic data provided insights in the downstream functional components of the drought response. The abscisic acid- and environmental stress-inducible protein TAS14 was highly induced by severe drought in potato and acts as a reliable biomarker for the level of stress perceived by the plant. The systems genetics approach supported a role for multiple genes responsive to severe drought stress of Solanum tuberosum. The combination of gene regulatory networks, expression quantitative trait loci mapping and phenotypic analysis proved useful for candidate gene selection.

Early blight of potato and tomato is caused by Alternaria fungi and negatively impacts crop yields. Environmental factors and plant maturity influence disease development, which is usually kept under control by fungicide applications. Wild tuber-bearing Solanum section Petota species are a promising source of resistance to early blight that could be used to control the disease, for example by crossbreeding or modern breeding approaches. An efficient screening method is a first prerequisite for the identification of resistant genotypes in wild Solanum germplasm. Here, we describe a protocol that can be used to rapidly screen for resistance to early blight in wild Solanum collections. This protocol provides a good starting point for the identification of resistant genotypes and is a step towards breeding for resistance to early blight using wild Solanum species.

Drought sensitivity of potato leads to a reduction in total tuber yield and marketable yield. An investigation of drought effects on tuber yield attributes will facilitate our understanding of how to reduce such huge yield losses. We have evaluated tuber yield, tuber size distribution and marketable yield of a set of 103 European commercial potato cultivars under irrigated and non-irrigated conditions in the field. The multi-year results from two locations, Connantre, France (2013–2015) and Nieuw-Namen in Zeeland, The Netherlands (2013–2014), were analysed. We used Normal and Gamma Distribution models to describe the tuber size distribution of tuber fresh weight and tuber number, respectively. The interactions among parameters of tuber size distribution and total/marketable tuber yield traits were analysed using correlation matrices and biplots. Finally, we used a 14K Infinium SNP marker array to find associations between the parameters or traits and genetic loci on the potato genome. Late foliage maturity facilitated a wider spread of tuber size distribution in favour of larger-sized tubers. Drought effects on total yield were representative of their impact on marketable yield, however, absolute values of total tuber number may not be indicative of marketable number of tubers. We found significant marker-trait associations between a region on chromosome 3 and the spread of tuber number distribution, size class with maximum tuber number and marketable fractions of tuber number and tuber weight. These findings will contribute to improvement and selection for drought tolerance in potato.

Cultivation of spinach requires high amounts of nitrogen (N), which puts a strain on the environment. A sustainable solution to this problem is to breed for crops with higher N use efficiency (NUE). The aim of this study was to provide tools for molecular breeding and to elucidate the genetic variation of factors contributing to NUE in spinach. A cross was made between two F1 hybrid cultivars contrasting in NUE. Several F1 progeny were self-pollinated and based on evaluation of the F2 generation, a mapping F2 population (335 individuals) of a single F1 was selected. SNP markers for the genetic map were discovered by RNA sequencing of the two parent cultivars, and 283 SNP markers were used to produce a genetic map comprising of six linkage groups (P01–P06), ranging in size from 46 to 116 cM. NUE related traits were determined for a set of F2:3 families grown under low and high N conditions in a hydroponics system under an Ingestad N-addition model. Interval mapping analysis detected 39 trait-specific QTLs, with several QTLs accumulating on P01 and P02 of the linkage map. The QTLs and in particular the P01 and P02 regions provide potential targets for the improvement of NUE in spinach.

Potato is an important food crop with high yields. However when exposed to drought it suffers major yield losses. Considering its global importance and the increasing incidence of drought due to climate change, research toward drought tolerance in potato remains imperative. We have studied a set of 103 commercial cultivars representing the genetic diversity in the European potato market. The cultivars were grown in different field locations in three subsequent years (2013–2015). Our aim was to understand how different field drought regimes affect canopy growth in potato, and how these effects translate to tuber yield. The field environmental conditions were monitored, and pictures of canopy ground cover during the growing season were taken. Canopy growth parameters were extracted by an iterative method using the beta sigmoid growth function to model canopy growth. At harvest, tuber yield was scored and tuber size was graded. The GGE (Genotype and Genotype-by-Environment) bi-plot and Finlay Wilkinson’s Regression were used to investigate Genotype x Environment interactions. We observed that the timing of the drought occurrence differentially affected canopy growth and tuber yield. Under drought stress, fast attainment of exponential growth and maximum canopy cover had negative effects on tuber formation and tuber bulking. Growth rate, maximum canopy cover, and area under the canopy curve (photosynthetic capacity over the growth season) were more important for tuber bulking than they were for tuber formation under drought stress. Cultivars with high yield were identified as potential material for improvement to drought tolerance. These findings will contribute to the breeding for drought-tolerant potato amidst the threats of climate change.

Genetics, biochemistry, and crop modelling are independently evolving disciplines; however, they complement each other in addressing some of the important challenges that crop science faces. One of these challenges is to improve our understanding of crop genotype-to-phenotype relationships in order to assist the development of high-yielding and resource-use efficient genotypes that can adapt to particular (future) target environments. Crop models are successful in predicting the impact of environmental changes on crop productivity. However, when critically tested against real experimental data, crop models have been shown to be less successful in predicting the impact of genotypic variation and genotype-by-environment interactions exhibited in genetic populations. In order to better model gene-trait-crop performance relationships in support of breeding and genetic engineering programmes, crop models need to be improved in terms of both model parameters and model structure. We argue that integration of quantitative genetics and photosynthesis biochemistry with modelling is a first step towards a new generation of improved crop models. With genetic information and biochemical understanding incorporated, crop modelling also generates new insights and concepts that can in turn be used to improve genetic analysis and biochemical modelling of complex traits. This modelling-genetics-biochemistry framework (the MGB triangle framework) stresses the synergy among the three disciplines, and may best serve as a step to achieve the ultimate goal of the more broadly framed “Crop Systems Biology” approach to improve efficiency of both classical breeding and genetic engineering programmes.

Abiotic and biotic stress factors are the major constrains for the realization of crop yield potential. As climate change progresses, the spread and intensity of abiotic as well as biotic stressors is expected to increase, with increased probability of crops being exposed to both types of stress. Shielding crops from combinatorial stress requires a better understanding of the plant’s response and its genetic architecture. In this study, we evaluated resistance to salt stress, powdery mildew and to both stresses combined in tomato, using the Solanum habrochaites LYC4 introgression line (IL) population. The IL population segregated for both salt stress tolerance and powdery mildew resistance. Using SNP array marker data, QTLs were identified for salt tolerance as well as Na+ and Cl− accumulation. Salt stress increased the susceptibility of the population to powdery mildew in an additive manner. Phenotypic variation for disease resistance was reduced under combined stress as indicated by the coefficient of variation. No correlation was found between disease resistance and Na+ and Cl− accumulation under combined stress Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance. These findings increase our understanding of the genetic regulation of responses to abiotic and biotic stress combinations and can provide leads to more efficiently breeding tomatoes and other crops with a high level of disease resistance while maintaining their performance in combination with abiotic stress.

Crop plants are subjected to a variety of stresses during their lifecycle, including abiotic stress factors such as salinity and biotic stress factors such as pathogens. Plants have developed a multitude of defense and adaptation responses to these stress factors. In the field, different stress factors mostly occur concurrently resulting in a new state of stress, the combined stress. There is evidence that plant resistance to pathogens can be attenuated or enhanced by abiotic stress factors. With stress tolerance research being mostly focused on plant responses to individual stresses, the understanding of a plant's ability to adapt to combined stresses is limited. In the last few years, we studied powdery mildew resistance under salt stress conditions in the model crop plant tomato with the aim to understand the requirements to achieve plant resilience to a wider array of combined abiotic and biotic stress combinations. We uncovered specific responses of tomato plants to combined salinity-pathogen stress, which varied with salinity intensity and plant resistance genes. Moreover, hormones, with their complex regulation and cross-talk, were shown to play a key role in the adaptation of tomato plants to the combined stress. In this review, we attempt to understand the complexity of plant responses to abiotic and biotic stress combinations, with a focus on tomato responses (genetic control and cross-talk of signaling pathways) to combined salinity and pathogen stresses. Further, we provide recommendations on how to design novel strategies for breeding crops with a sustained performance under diverse environmental conditions.

High affinity potassium transporters (HKT) are recognized as important genes for crop salt tolerance improvement. In this study, we investigated HvHKT1;5 as a candidate gene for a previously discovered quantitative trait locus that controls shoot Na+ and Na+/K+ ratio in salt-stressed barley lines on a hydroponic system. Two major haplotype groups could be distinguished for this gene in a barley collection of 95 genotypes based on the presence of three intronic insertions; a designated haplotype group A (HGA, same as reference sequence) and haplotype group B (HGB, with insertions). HGB was associated with a much stronger root expression of HKT1;5 compared to HGA, and consequently higher K+ and lower Na+ and Cl− concentrations and a lower Na+/K+ ratio in the shoots three weeks after exposure to 200 mM NaCl. Our experimental results suggest that allelic variation in the promoter region of the HGB gene is linked to the three insertions may be responsible for the observed increase in expression of HvHKT1;5 alleles after one week of salt stress induction. This study shows that in barley - similar to wheat and rice - HKT1;5 is an important contributor to natural variation in shoot Na+ exclusion.

Spinach is a leafy vegetable that requires a high N fertilization to have a satisfactory yield and quality, in part because it has poor nitrogen use efficiency (NUE). Therefore, there is a need to breed for cultivars with an excellent NUE. To this end the genetic diversity for NUE-related traits was studied in a diverse set of commercial cultivars. This set was evaluated in a hydroponic system using the Ingestad model; the system was set at a relative growth rate of 0.14 and 0.18 g g-1 day-1 (low and high N, respectively). Experiments were performed at low and high plant density. Traits monitored for single plants included fresh and dry weight, leaf area, specific leaf area, dry weight ratio between root and shoot, and chlorophyll content. The high density experiment showed more genotypic variation for the observed traits than the low density one. Biomass production was considerably lower at low than at high N. Path analysis revealed that leaf area had the highest direct effect on NUE, while specific leaf area was an important trait determining variation in NUE at low N. Slow and fast growing genotypes were shown to use different strategies to utilize N, and these strategies are expressed differently at high and low N availability. This indicates that improving spinach for NUE is feasible using the analysed genotypes as source material, and different strategies can be targeted for adaptation of spinach cultivars to low N conditions.

Gewassen beïnvloeden de bodem waarop ze geteeld worden. In deze verkennende literatuurstudie is onderzocht wat er bekend is over variatie tussen verschillende genotypen/plantenrassen met betrekking tot hun effect op bodemkwaliteit voor een aantal ruwvoedergewassen, in het bijzonder Maïs (Zea mays), Engels raaigras (Lolium perenne) en Witte klaver (Trifolium repens). Dergelijke variatie zou mogelijkheden kunnen bieden om via veredeling en het inzetten van specifieke rassen de bodemkwaliteit te verbeteren. Er is betrekkelijk weinig onderzoek vanuit deze invalshoek gedaan. De focus van de studie was gericht op variatie in wortelarchitectuur, en wortelexudatie en wortelafsterving/nieuwvorming in relatie tot het organische stof gehalte en beschikbaarheid van nutriënten, en de bodem biota, met name mycorrhiza.

Miscanthus is a woody rhizomatous C4 grass that can be used as a CO2 neutral biofuel resource. It has potential to grow in marginal areas such as saline soils, avoiding competition for arable lands with food crops. This study explored genetic diversity for salt tolerance in Miscanthus and discovered mechanisms and traits that can be used to improve the yield under salt stress. Seventy genotypes of Miscanthus (including 57 M. sinensis, 5 M. sacchariflorus, and 8 hybrids) were evaluated for salt tolerance under saline (150 mM NaCl) and normal growing conditions using a hydroponic system. Analyses of shoot growth traits and ion concentrations revealed the existence of large variation for salt tolerance in the genotypes. We identified genotypes with potential for high biomass production both under control and saline conditions that may be utilized for growth under marginal, saline conditions. Several relatively salt tolerant genotypes had clearly lower Na+ concentrations and showed relatively high K+/Na+ ratios in the shoots under salt stress, indicating that a Na+ exclusion mechanism was utilized to prevent Na+ accumulation in the leaves. Other genotypes showed limited reduction in leaf expansion and growth rate under saline conditions, which may be indicative of osmotic stress tolerance. The genotypes demonstrating potentially different salt tolerance mechanisms can serve as starting material for breeding programs aimed at improving salinity tolerance of Miscanthus.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic variation that can be used as molecular markers. The SNPs that are hidden in sequence databases can be unlocked using bioinformatic tools. For efficient application of these SNPs, the sequence set should be error-free as much as possible, targeting single loci and suitable for the SNP scoring platform of choice. We have developed a pipeline to effectively mine SNPs from public EST databases with or without quality information using QualitySNP software, select reliable SNP and prepare the loci for analysis on the Illumina GoldenGate genotyping platform. The applicability of the pipeline was demonstrated using publicly available potato EST data, genotyping individuals from two diploid mapping populations and subsequently mapping the SNP markers (putative genes) in both populations. Over 7000 reliable SNPs were identified that met the criteria for genotyping on the GoldenGate platform. Of the 384 SNPs on the SNP array approximately 12% dropped out. For the two potato mapping populations 165 and 185 SNPs segregating SNP loci could be mapped on the respective genetic maps, illustrating the effectiveness of our pipeline for SNP selection and validation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-009-9377-5) contains supplementary material, which is available to authorized users.

NBS profiling is a method for the identification of resistance gene analog (RGA) derived fragments. Here we report the use of NBS profiling for the genome wide mapping of RGA loci in potato. NBS profiling analyses on a minimal set of F1 genotypes of the diploid mapping population previously used to generate the ultra dense (UHD) genetic map of potato, allowed us to efficiently map polymorphic RGA fragments relative to 10,000 existing AFLP markers. In total, 34 RGA loci were mapped, of which only 13 contained RGA sequences homologous to RGAs genetically positioned at approximately similar positions in potato or tomato. The remaining RGA loci mapped either at approximate chromosomal regions previously shown to contain RGAs in potato or tomato without sharing homology to these RGAs, or mapped at positions not yet identified as RGA-containing regions. In addition to markers representing RGAs with unknown functions, segregating markers were detected that were closely linked to four functional R genes that segregate in the UHD mapping population. To explore the potential of NBS profiling in RGA transcription analyses, RNA isolated from different tissues was used as template for NBS profiling. Of all the fragments amplified approximately 15% showed putative intensity or absent/present differences between different tissues suggesting putative tissue specific RGA or R gene transcription. Putative absent/present differences between individuals were also found. In addition to being a powerful tool for generating candidate gene markers linked to R gene loci, NBS profiling, when applied to cDNA, can be instrumental in identifying those members of an R gene cluster that are transcribed, and thus putatively functional.

Systematic relationships are important criteria for researchers and breeders to select materials. We evaluated a novel molecular technique, nucleotide binding site (NBS) profiling, for its potential in phylogeny reconstruction. NBS profiling produces multiple markers in resistance genes and their analogs (RGAs). Potato (Solanum tuberosum L.) is a crop with a large secondary genepool, which contains many important traits that can be exploited in breeding programs. In this study we used a set of over 100 genebank accessions, representing 49 tuber-bearing wild and cultivated Solanum species. NBS profiling was compared to amplified fragment length polymorphism (AFLP). Cladistic and phenetic analyses showed that the two techniques had similar resolving power and delivered trees with a similar topology. However, the different statistical tests used to demonstrate congruency of the trees were inconclusive. Visual inspection of the trees showed that, especially at the lower level, many accessions grouped together in the same way in both trees; at the higher level, when looking at the more basal nodes, only a few groups were well supported. Again this was similar for both techniques. The observation that higher level groups were poorly supported might be due to the nature of the material and the way the species evolved. The similarity of the NBS and AFLP results indicate that the role of disease resistance in speciation is limited.

Phosphorus (P) is often an important limiting factor for crop yields, but rock phosphate as fertilizer is a non-renewable resource and expected to become scarce in the future. High P input levels in agriculture have led to environmental problems. One of the ways to tackle these issues simultaneously is improving phosphorus use efficiency (PUE) of the crops through breeding. In this review, we describe plant architectural and physiological traits important for PUE. Subsequently, we discuss efficient methods of screening for PUE traits. We address targeted cultivation methods, including solid and hydroponic systems, as well as testing methods, such as image analysis systems, and biomass and photosynthesis measurements. Genetic variation for PUE traits has been assessed in many crops, and genetics of PUE has been studied by quantitative trait loci (QTL) analyses and genome-wide association study. A number of genes involved in the plant’s response to low P have been characterized. These genes include transcription factors, and genes involved in signal transduction, hormonal pathways, sugar signalling, P saving metabolic pathways, and in P scavenging, including transporters and metabolites and/or ATP-ases mobilizing P in the soil. In addition, the role of microorganisms promoting PUE of plants, particularly arbuscular mycorrhizal fungi is discussed. An overview is given of methods for selecting for optimal combinations of plant and fungal genotypes, and their genetics, incl. QTLs and genes involved. In conclusion, significant progress has been made in selecting for traits for PUE, developing systems for the difficult but highly relevant root phenotyping, and in identifying QTLs and genes involved.

Background Within onion, Allium cepa L., the availability of disease resistance is limited. The identification of sources of resistance in related species, such as Allium roylei and Allium fistulosum, was a first step towards the improvement of onion cultivars by breeding. SNP markers linked to resistance and polymorphic between these related species and onion cultivars are a valuable tool to efficiently introgress disease resistance genes. In this paper we describe the identification and validation of SNP markers valuable for onion breeding. Results Transcriptome sequencing resulted in 192 million RNA seq reads from the interspecific F1 hybrid between A. roylei and A. fistulosum (RF) and nine onion cultivars. After assembly, reliable SNPs were discovered in about 36 % of the contigs. For genotyping of the interspecific three-way cross population, derived from a cross between an onion cultivar and the RF (CCxRF), 1100 SNPs that are polymorphic in RF and monomorphic in the onion cultivars (RF SNPs) were selected for the development of KASP assays. A molecular linkage map based on 667 RF-SNP markers was constructed for CCxRF. In addition, KASP assays were developed for 1600 onion-SNPs (SNPs polymorphic among onion cultivars). A second linkage map was constructed for an F2 of onion x A. roylei (F2(CxR)) that consisted of 182 onion-SNPs and 119 RF-SNPs, and 76 previously mapped markers. Markers co-segregating in both the F2(CxR) and the CCxRF population were used to assign the linkage groups of RF to onion chromosomes. To validate usefulness of these SNP markers, QTL mapping was applied in the CCxRF population that segregates for resistance to Botrytis squamosa and resulted in a QTL for resistance on chromosome 6 of A. roylei. Conclusions Our research has more than doubled the publicly available marker sequences of expressed onion genes and two onion-related species. It resulted in a detailed genetic map for the interspecific CCxRF population. This is the first paper that reports the detection of a QTL for resistance to B. squamosa in A. roylei. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0879-0) contains supplementary material, which is available to authorized users.

Breeding for stress-resilient crops strongly depends on technological and biological advancements that have provided a wealth of information on genetic variants and their contribution to stress tolerance. In the context of the upcoming challenges for agriculture due to climate change, such as prolonged and/or increased stress intensities, CO2 increase and stress combinations, hierarchizing this information is key to accelerating crop improvement towards sustained or even increased productivity. We propose traits with high scalability to yield and crop performance that can be targeted for improvement and provide examples of recent discoveries with potential applicability in breeding. Critical to success is the integrated analysis of the phenotypes of genetic variants across different environmental variables using modelling approaches and high-throughput phenotyping.

Highlight Salt stress severity differentially affected partial powdery mildew resistance. Stress combination uniquely resulted in accelerated senescence. mlo-based and R-gene-mediated resistance maintained robustness across salt treatments., Stress conditions in agricultural ecosystems can occur at variable intensities. Different resistance mechanisms against abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100, and 150mM NaCl) on powdery mildew resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as near-isogenic lines (NILs) carrying the resistance gene Ol-1 (associated with a slow hypersensitivity response; HR), ol-2 (an mlo mutant associated with papilla formation), and Ol-4 (an R gene associated with a fast HR). Powdery mildew resistance was affected by salt stress in a genotype- and stress intensity-dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied by accelerated cell death-like senescence. In contrast, severe salt stress (150mM) reduced disease symptoms. Na+ and Cl− accumulation in the leaves was linearly related to the decreased pathogen symptoms under severe stress. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress in NIL-Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes and the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant’s performance under the combination of abiotic and biotic stress.

In plants, tolerance to drought stress is a result of numerous minor effect loci in which transcriptional regulation contributes significantly to the observed phenotypes. Under severe drought conditions, a major expression quantitative trait loci hotspot was identified on chromosome five in potato. A putative Nuclear factor y subunit C4 was identified as key candidate in the regulatory cascade in response to drought. Further investigation of the eQTL hotspots suggests a role for a putative Homeobox leucine zipper protein 12 in relation to drought in potato. Genes strongly co-expressed with Homeobox leucine zipper protein 12 were plant growth regulators responsive to water deficit stress in Arabidopsis thaliana, implying a possible conserved mechanism. Integrative analysis of genetic, genomic, phenotypic and transcriptomic data provided insights in the downstream functional components of the drought response. The abscisic acid- and environmental stress-inducible protein TAS14 was highly induced by severe drought in potato and acts as a reliable biomarker for the level of stress perceived by the plant. The systems genetics approach supported a role for multiple genes responsive to severe drought stress of Solanum tuberosum. The combination of gene regulatory networks, expression quantitative trait loci mapping and phenotypic analysis proved useful for candidate gene selection.

Molecular markers are effective tools to investigate genetic diversity for resistance to pathogens. NBS (nucleotide-binding site) profiling is a PCR (polymerase chain reaction)-based approach to studying genetic variability that specifically targets chromosome regions containing R-genes and R-gene analogues. We used NBS profiling to measure genetic diversity among 58 accessions of durum wheat. Mean polymorphism rates detected using MseI and AluI as restriction enzymes were 34% and 22%, respectively. Mean number of polymorphisms per enzyme–primer combination was equal to 23.8 ± 5.9, ranging from 13 to 31 polymorphic bands. In total, 96 markers over 190 indicated a good capacity to discriminate between accessions (the polymorphic index content ranging from 0.30 to 0.50). The results obtained with NBS profiling were compared with simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) data of the same set of accessions. The genetic distances computed with 190 NBS profiling markers were in close agreement with those obtained with AFLP and SSR markers (r = 0.73 and 0.76, respectively). Our results indicate that NBS profiling provides an effective means to investigate genetic diversity in durum wheat.

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50-90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.

Nature 542, 307–312 (2017); doi:10.1038/nature21370 The Acknowledgements section of this Article should have included the following sentence: “Metabolite imaging was conducted at Metabolomics Australia (School of BioSciences, The University of Melbourne, Australia), a NCRIS initiative under Bioplatforms Australia Pty Ltd.

Plants growing in their natural habitats are often challenged simultaneously by multiple stress factors, both abiotic and biotic. Research has so far been limited to responses to individual stresses, and understanding of adaptation to combinatorial stress is limited, but indicative of non-additive interactions. Omics data analysis and functional characterization of individual genes has revealed a convergence of signaling pathways for abiotic and biotic stress adaptation. Taking into account that most data originate from imposition of individual stress factors, this review summarizes these findings in a physiological context, following the pathogenesis timeline and highlighting potential differential interactions occurring between abiotic and biotic stress signaling across the different cellular compartments and at the whole plant level. Potential effects of abiotic stress on resistance components such as extracellular receptor proteins, R-genes and systemic acquired resistance will be elaborated, as well as crosstalk at the levels of hormone, reactive oxygen species, and redox signaling. Breeding targets and strategies are proposed focusing on either manipulation and deployment of individual common regulators such as transcription factors or pyramiding of non- (negatively) interacting components such as R-genes with abiotic stress resistance genes. We propose that dissection of broad spectrum stress tolerance conferred by priming chemicals may provide an insight on stress cross regulation and additional candidate genes for improving crop performance under combined stress. Validation of the proposed strategies in lab and field experiments is a first step toward the goal of achieving tolerance to combinatorial stress in crops.

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.

Ion homeostasis is considered to be one of the most important mechanisms underlying salt stress tolerance. We used the Steptoe × Morex barley doubled haploid population to screen for genetic variation in response to salinity stress at an early development stage in a hydroponics system, focusing on ion homeostasis. Salinity induced a strong adverse effect on growth of the parents and their derived population, with Steptoe as the more tolerant parent. Steptoe maintained higher concentrations of K+, Na+ and Cl− in the roots and a similar shoot/root ion ratio (

Background: Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes. Results: In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach. Conclusions: In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.