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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.

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.

Ample nitrogen (N) is required for potato production, but its use efficiency is low. N supply strongly interacts with maturity type of the cultivar grown. We assessed whether variation among 189 cultivars grown with 75 or 185 kg available N/ha in 2 years would allow detecting quantitative trait loci (QTLs) for relevant traits. Using phenotypic data, we estimated various traits and carried out a genome-wide association study (GWAS) with kinship correction. Twenty-four traits and 10,747 markers based on single-nucleotide polymorphisms from a 20K Infinium array for 169 cultivars were combined in the analysis. N level affected most traits and their interrelations and influenced the detection of marker–trait associations, some were N-dependent, others were detected at both N levels. Ninety percent of the latter accumulated on a hotspot on Chromosome 5. Chromosomes 2 and 4 also contained regions with multiple associations. After correcting for maturity, the number of QTLs detected was much lower, especially of those common to both N levels, however, interestingly, the region on Chromosome 2 accumulated several QTLs. There is scope for marker-assisted selection for maturity, with the main purpose of improving characteristics within a narrow range of maturity types, in order to break the strong links between maturity type and traits like N use efficiency.

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.

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.

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.

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.

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.

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.

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.

A spring barley collection of 192 genotypes from a wide geographical range was used to identify quantitative trait loci (QTLs) for salt tolerance traits by means of an association mapping approach using a thousand SNP marker set. Linkage disequilibrium (LD) decay was found with marker distances spanning 2-8 cM depending on the methods used to account for population structure and genetic relatedness between genotypes. The association panel showed large variation for traits that were highly heritable under salt stress, including biomass production, chlorophyll content, plant height, tiller number, leaf senescence and shoot Na(+), shoot Cl(-) and shoot, root Na(+)/K(+) contents. The significant correlations between these traits and salt tolerance (defined as the biomass produced under salt stress relative to the biomass produced under control conditions) indicate that these traits contribute to (components of) salt tolerance. Association mapping was performed using several methods to account for population structure and minimize false-positive associations. This resulted in the identification of a number of genomic regions that strongly influenced salt tolerance and ion homeostasis, with a major QTL controlling salt tolerance on chromosome 6H, and a strong QTL for ion contents on chromosome 4H.

Potato is the third most important staple food crop in terms of consumption, yet it is relatively susceptible to yield loss because of drought. As a first step towards improving drought tolerance in this crop, we set out to identify the genetic basis for drought tolerance in a diploid potato mapping population. Experiments were carried out under greenhouse conditions in two successive years by recording four physiological, seven growth and three yield parameters under stress and recovery treatments. Genotypes showed significant variation for drought and recovery responses. The traits measured had low to moderately high heritabilities (ranging from 22 to 74 %). A total of 47 quantitative trait loci (QTL) were identified, of which 28 were drought-specific, 17 under recovery treatment and two under well-watered conditions. The majority of these growth and yield QTL co-localized with a QTL for maturity on chromosome 5. Four QTL for δ(13)C, three for chlorophyll content and one for chlorophyll fluorescence (F(v)/F(m)) were found to co-localize with yield and other growth trait QTL identified on other chromosomes. Several multi-year and multi-treatment QTL were detected and QTL × environment interaction was found for δ(13)C. To our knowledge, this is the first comprehensive QTL study on water deficit and recovery potential in potato. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9728-5) contains supplementary material, which is available to authorized users.

Drought stress is a major abiotic constraint limiting crop production worldwide. Screening for drought tolerance and the traits that enhance drought tolerance is not straightforward in large mapping populations. In this study, we investigated the possibility of screening a mapping population in vitro for PEG-induced water deficit stress and recovery potential. We have measured several shoot and root growth parameters or traits in the C × E diploid potato mapping population. Significant variation was observed for genotype-specific responses to water deficit and recovery potential. Genetic variation and heritability estimates were high to very high for the measured traits depending on growth conditions. In order to identify potato QTLs for drought tolerance and recovery potential an SNP marker-rich integrated linkage map was used. A total of 23 QTLs were detected under control, stress and recovery treatments explaining 10.3–22.4% of the variance for each phenotypic trait. Among these, 10 QTLs were located on chromosome 2. Three QTLs involved in the important trait root to shoot ratio were identified on linkage groups 2, 3 and 8. These loci explained together 41.1% of the variance for this trait, and may be breeding targets for stress tolerance and yield in the field as well. The SNP markers derived from EST sequences underlying these QTLs led to the identification of putative candidate genes for further study in potato. This study constitutes the first knowledge of in vitro screening of a mapping population for drought tolerance in potato.

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato. Electronic supplementary material The online version of this article (doi:10.1007/s00122-011-1602-z) contains supplementary material, which is available to authorized users.

Background: Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance. Methodology/Principal Findings: The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers. Conclusions/Significance: We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance. © 2010 González et al.

Background Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs. Results We have developed PolySSR, a new pipeline to identify polymorphic SSRs rather than just SSRs. Sequence information is obtained from public EST databases derived from heterozygous individuals and/or at least two different genotypes. The pipeline includes PCR-primer design for the putatively polymorphic SSR markers, taking into account Single Nucleotide Polymorphisms (SNPs) in the flanking regions, thereby improving the success rate of the potential markers. A large number of polymorphic SSRs were identified using publicly available EST sequences of potato, tomato, rice, Arabidopsis, Brassica and chicken. The SSRs obtained were divided into long and short based on the number of times the motif was repeated. Surprisingly, the frequency of polymorphic SSRs was much higher in the short SSRs. Conclusion PolySSR is a very effective tool to identify polymorphic SSRs. Using PolySSR, several hundred putative markers were developed and stored in a searchable database. Validation experiments showed that almost all markers that were indicated as putatively polymorphic by polySSR were indeed polymorphic. This greatly improves the efficiency of marker development, especially in species where there are low levels of polymorphism, like tomato. When combined with the new sequencing technologies PolySSR will have a big impact on the development of polymorphic SSRs in any species. PolySSR and the polymorphic SSR marker database are available from http://www.bioinformatics.nl/tools/polyssr/.

Molecular markers based upon a novel lettuce LTR retrotransposon and the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of disease resistance-associated genes have been combined with AFLP markers to generate a 458 locus genetic linkage map for lettuce. A total of 187 retrotransposon-specific SSAP markers, 29 NBS-LRR markers and 242 AFLP markers were mapped in an F2 population, derived from an interspecific cross between a Lactuca sativa cultivar commonly used in Europe and a wild Lactuca serriola isolate from Northern Europe. The cross has been designed to aid efforts to assess gene flow from cultivated lettuce into the wild in the perspective of genetic modification biosafety. The markers were mapped in nine major and one minor linkage groups spanning 1,266.1 cM, with an average distance of 2.8 cM between adjacent mapped markers. The markers are well distributed throughout the lettuce genome, with limited clustering of different marker types. Seventy-seven of the AFLP markers have been mapped previously and cross-comparison shows that the map from this study corresponds well with the previous linkage map.

Spinach (Spinacia oleracea L.) is one of the most consumed leafy vegetables worldwide and it is considered to be highly nutritious. Spinach is a short-cycle leafy crop that has a high demand for nitrogen in order to rapidly come to a harvestable product that has the required dark green colour within a reasonable harvest window. In commercial production of spinach the recovery of N is poor, which may result in environmental pollution. To increase sustainability of both organic and conventional spinach cultivation there is a need to reduce the dependency on high levels of nitrogen. Growers therefore urgently need cultivars with a satisfactory yield under reduced N input conditions. Nitrogen use efficiency (NUE), defined as the ability to produce high biomass per unit N applied, is low in spinach. The present study aims to evaluate spinach genotypes for selectable traits under varying N supply and provide tools and knowledge to facilitate the development of varieties with good yield, quality and stability under low N input. To minimise environmental variation affecting the identification of traits related to NUE a screening method was developed using a hydroponics system. The genetic diversity for NUE related traits was first studied with 24 commercial cultivars under contrasting levels of N supply based on the Ingestad model with a steady-state N application. This demonstrated that the hydroponics screening strategy as a pre-screening tool enabled reliable detection of heritable variation among cultivars for NUE-related traits under optimal as well as suboptimal N input. Shoot dry weight and leaf area were preferred selectable traits for the detection of heritable differences contributing to NUE in spinach. The effect of N application strategy was examined in seven cultivars grown under hydroponics conditions with low and high N levels supplied either as a single bulk N application resembling N fertilization in field cultivation, or a steady-state N application according to Ingestad. The latter application strategy provided more stable and reproducible conditions for determination of genetic differences in NUE under low N conditions for a short-cycle leafy vegetable crop. Several tools for molecular genetic evaluation of NUE in spinach were provided as well, including a SNP marker set for marker-assisted breeding, a genetic mapping population with a corresponding genetic map, and the identification of two major QTL regions contributing to growth under low N conditions. With these tools, an efficient strategy for breeding for NUE efficiency in spinach would include screening under controlled conditions at high and low N using leaf area, biomass and root to shoot ratio as selectable traits, and QTL identification of genetic factors that can be targeted and combined using marker-assisted selection. An in depth genotype by environment interaction analysis using six field trials showed that environmental factors like temperature, soil, and management strongly influence nitrogen availability in the soil in a short cycle crop like spinach. This severely complicates selection and breeding for NUE of spinach under field conditions, and emphasizes the importance of performing trials under better controllable conditions for genetic dissection of NUE and discovery of genetic factors contributing to NUE. It also underscores the importance of validating these findings in various field trials. Multi-environment field trials with different levels of N fertilization will then allow selection of cultivars that combine stable performance under various low input growing conditions with high yields under more favorable conditions.

Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited. In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies. In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance. Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress. To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects. The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control. Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

Salinity is the most severe abiotic stress perceived by plants and affects about 800 million hectares of land worldwide, including 20% of the world’s highly productive irrigated land. Significant crop yield losses are observed due to salinity. Salinization is increasing because of poor irrigation management and climate change. Improving salt tolerance in crops is for these reasons an important target for plant breeding in the near future. However, salinity tolerance in plants is not easy to breed for due to its interaction with many physiological processes controlled by many genes and their interaction with the environment. Barley is a good model crop to study different mechanisms conferring salt tolerance in cereals. A traditional QTL mapping approach in combination with a new association mapping method allowed us to efficiently explore the genetics and genetic diversity of salt tolerance in barley. Improvements of the association mapping technology highly increased detection power and mapping accuracy. The traits and QTLs identified in this thesis point out both osmotic and ionic stress tolerant genes as important targets for salt tolerance breeding. This thesis provides tools to plant breeders for the application of marker-assisted introgression breeding of salt tolerance genes in their breeding programs. Some QTLs were found to be syntenic with the important QTLs/genes for salt tolerance found in wheat and rice such as Na+ and K+ transporter gene families. Other QTLs were new and suggest the presence of novel genes that play an important role in plant ion homeostasis, transportation of Cl- and Ca2+ and osmotic tolerance. We demonstrated that association mapping can be a powerful approach to dissect the complexity of salt tolerance in barley. The newly available high-density SNP map of barley and the barley genome sequence in the near future further increases the accuracy of mapping studieswith the association panel and will greatly facilitate the cloning of the genes underlying salt tolerance in barley. This thesis thus contributes to better a understanding of the physiological and genetic basis of salt tolerance and improved breeding strategies for the development of salt tolerant varieties.

Drought is the most important cause of crop and yield loss around the world. Breeding for drought tolerance is not straightforward, as drought is a complex trait. A better understanding of the expression of drought traits, the genes underlying the traits and the way these genes interact will significantly increase the success of breeding for drought tolerance. Potato is an important food crop, yet it is relatively susceptible to drought. As a first step towards identifying the genetic basis for drought tolerance in potato, we make use of diploid potato populations that have been genetically well characterized (CxE, SHxRH). The CxE population was extensively evaluated for drought tolerance in vitro and for two successive years (2008, 2009) under greenhouse conditions and the data were used for QTL mapping. For optimal QTL mapping, we expanded the CxE and SHxRH genetic maps with 499 SNP markers (two arrays 384 and 768SNP arrays respectively, enriched for putative stress tolerance candidate genes). The SNPs were discovered in public EST databases using QualitySNP software and detected with the Illumina GoldenGate assay. About 300 SNPs served as bridge markers between the CxE and SHxRH maps. This will enable us to make use of the extensive genetic and sequence information of the SHxRH population and the RH genome sequence. With the availability of the potato genome sequence of the doubled monoploid DM1-3 516R44 (DM) (www.potatogenome.net), it was possible to further examine the SNP marker loci for paralogs and intron spanning sequences. In total 732 SNP marker loci were found to be unique in the potato genome sequence. Many of these SNP markers not only served as landmarks on the genetic map but may also as putative genes underlying quantitative traits. In addition the validated SNP markers are now utilized as anchors in the potato physical map. We investigated the possibility of screening potato for relevant drought traits in in vitro cultures and evaluated the CxE population for the response to PEG-induced water deficit stress and recovery potential after stress. Significant genetic variation was observed for the response to drought and for recovery potential. Several shoot and root growth traits were measured. In this study the genetic variation and heritability estimates were high to very high for the measured traits under control and recovery condition. In total 23 QTLs were detected in plants under control, stress and recovery treatments. Interesting putative candidate genes that may underly stress response QTLs were identified. The drought tolerance evaluation of the CxE population in pots in the greenhouse included traits like leaf Relative Water Content, δ13C as a measure of Water Use Efficiency, Chlorophyll Fluorescence, Chlorophyll Content, shoot and root biomass and tuber yield. The progeny displayed a wide contrast for drought tolerance, with individuals surviving and recovering completely after 3 weeks of drought, and others completely wilted beyond recovery. Most of the traits had high heritabilities. QTLs effective in multiple treatments and years were detected for tuber number, tuber weight, plant height, shoot fresh and dry weight. Other QTLs were found to be dependent on the environment: QTL x Environment interaction was found for leaf d13C under drought conditions and we speculate that the function of δ13C was genetically split into a stomatal and non-stomatal component. Many of the QTLs for growth traits measured both in the greenhouse and in in vitro cultures were specific to either of the growth conditions. Yet significant QTLs that were detected for plant height, shoot dry weight, fresh biomass for plants grown in the greenhouse were also found when the population was grown in vitro. These QTLs may be less affected by environmental influences, and we may therefore expect that some of these QTLs will be relevant under field conditions as well. This also suggests that the in vitro system may be used for preliminary selection in breeding programmes for specific performance-related traits. The genetic architecture of transcript-level variation for drought response was captured in the potato population CxE and mapped as expression QTLs (eQTLs). We anchored the differentially expressed genes to the genome sequence of potato, and this enabled us to determine whether the transcription of these genes (the eQTLs) is in cis or in trans regulated. The combined use of genome-wide detection of eQTLs in combination with genome sequence information for gene location has enables us to detect regulatory hot spots for drought response in the CxE population. Based on gene ontology annotation, a number of eQTLs were detected for genes known to be involved in drought signal transduction and drought-induced transcriptional regulation, and for redox genes. Examination of co-localization of eQTLs and phenotypic QTLs identified several interesting eQTLs for genes that may be involved in specifying the phenotypic QTL, for instance, the eQTL for a gene that was annotated with a putative function in the photosystem II light reaction colocalized with trait QTL of chlorophyll florescence (Fv/Fm) on chromosome 1, along with other genes involved in 139 drought response such as heat shock proteins and signaling proteins with known induced expression under stress conditions. On chromosome 10, eQTLs for genes involved in carbon partitioning, signaling receptor kinases, transcription factors and hormone and lipid metabolism were colocalized with phenotypic QTLs for chlorophyll content and stomatal component of δ13C. As we have only touched the surface of the information contained in the transcriptome dataset combined with the phenotyping data, continued efforts on mining the dataset and in depth analysis will most likely reveal more putative candidate genes for QTL effects. This thesis constitutes the first knowledge of in vitro and greenhouse screening for drought tolerance in potato and has led to the description of important traits for screening and selection in breeding for drought tolerance. The QTLs identified in this thesis may be interesting targets for potato breeding to improve drought tolerance of the potato crop. Furthermore, our results illustrate the power of application of integrated genetic and genomics approaches to unravel the molecular components underlying abiotic stress tolerance traits.

Cowpea [Vigna unguiculata (L.) Walp.] is a most versatile African crop, it feeds people, their livestock and because of its ability in nitrogen-fixation, it improves soil fertility, and consequently helps to increase the yields of cereal crops when grown in rotation and contributes to the sustainability of cropping systems. Because of its ability to tolerate some level of drought stress, cowpea is a crop of choice where > 10 million hectares are cultivated to cowpea in the semiarid Savanna and Sahelian zones of West and Central Africa. However due to the infrequent drought stress throughout cropping seasons over the last 3 decades, the crop suffers important yield reduction. Moreover, cowpea plants under water stress condition are more vulnerable to diseases, parasites and insect pests attacks. In this thesis we carried out genetic analyses of seedling and terminal drought tolerance and cowpea bacterial blight (CoBB) resistance. Two cowpea genotypes with contrasting reactions to drought and CoBB stresses, Danila (resistant) and TVu7778 (susceptible) were used to develop recombinant inbred lines (RILs) mapping population. The RILs and parents were phenotyped for CoBB resistance and for physiological drought tolerance traits and productivity traits under different water regimes in multiple environments at seedling and adult plant stages. A genetic linkage map of 282 single nucleotide polymorphism (SNP) loci covering a map distance of 633 cM distributed over 11 linkage groups (LG) from the same RILs was used for quantitative traits loci (QTL) analyses. Moisture stress significantly affected RILs performances with number of pods per plant as the yield component most adversely affected by water stress. Correlation and path analyses revealed that grain yield components (mainly number of pods per plant) and plant biomass had the largest direct effects on grain yield under moisture stress and irrigation. Stem greenness was an excellent predictor of seedling survival to drought (r2 = 0.91). Two QTLs were identified for each of the three traits scored under greenhouse, drought-induced trifoliate senescence (DTS), stem greenness (Stg) and survival (Sur) on LG3 and LG7. For all traits measured under field trials, a total of 42 QTLs were detected, 4 for stomatal conductance (Gs), 6 for delayed leaf senescence (DLS), 5 for flowering time and 16 for grain yield components (pod number/plant, seed number/pod, seed weight), 6 for grain yield and 5 for fodder yield. Three QTLs were detected for CoBB resistance, with two major ones (named CoBB-1 and CoBB-2 confirmed over two experiments) on LG3 and LG5 and one minor QTL (CoBB-3 only for experiment 1) on LG9. Although number of QTLs detected seems to be important, association between QTLs of different traits sharing a common genomic region was observed on LG3, LG5, LG7 and LG8 where QTLs for Gs, DLS, and flowering time co-localized with QTLs for yield parameters, DLS and DTS shared common chromosomal regions with CoBB resistance, on LG3 and LG5. These results suggest that common genes might mediate CoBB resistance and DLS. Two QTLs detected for DLS in the field, co-localized with QTLs under greenhouse for seedling drought tolerance traits DTS, Stg and Sur on LG7. This indicates the presence of potential loci controlling senescence in this genomic region. Moreover, this genomic region represents a syntenic genomic region between cowpea, soybean and Medicago. Our findings provide evidence for QTLs mediating seedling and terminal drought tolerance and CoBB resistance in cowpea. QTL and phenotypic analysis revealed that it should be possible to pyramid CoBB resistance with seedling and terminal drought tolerance. The fact that the genetic map of Danila x TVu7778 is integrated in a consensus map of cowpea of 6 RIL populations will permit comparative genomic studies which will enhance the discovery of functional markers for MAS of seedling and terminal drought tolerance and CoBB resistance in cowpea.