Expression Analysis of Auxin Regulated Genes in \u3cem\u3ePopulus\u3c/em\u3e

Due to its many advantageous characteristics, such as a small sequenced genome, ease of vegetative propagation and availability of genomic tools and databases, Populus is widely becoming accepted as the model species among trees. In addition, DOE has chosen hybrid poplar as the model bioenergy feedstock tree. Due to the growing importance of the Populus species, genetic and genomic resources (EST and BLAST databases, genetic maps, etc.) are becoming increasingly available and are leading to a greater understanding of the functionality of the Populus genome. The goal of this study was to use these resources to further characterize the genetic controls of root growth and development so that these mechanisms may eventually be manipulated to improve carbon sequestration ability in belowground sinks. Because auxin is known to play an important role in lateral root growth as well as many other aspects of plant development, a sequenced subtracted cDNA library from poplar was used to study the expression of genes up and down-regulated in response to exogenous auxin treatments. Results from this study indicate that a daily 9-day exogenous auxin treatment may have induced a stress response as indicated by the high percentage of WRKY transcription factors and stress related proteins that were up-regulated in response to the treatment regimen. A second study was also done using whole-genome oligonucleotide microarray technology to further analyze auxin regulated gene expression including Populus homologs of AUX/IAA and ARF genes in Arabidopsis. Results from this study did not appear to correlate well with real-time RT-PCR results indicating that, in the future, more reps need to be used to give the experiment the statistical power necessary to accurately find differentially expressed genes. Results from these gene expression studies can then be used to guide the development of poplar transgenics with increased root growth

Expression profiling of oocyte specific genes, transcription factors and microRNAs during early embryonic development in rainbow trout (Onchorhyncus mykiss)

Genes specifically expressed in oocytes are important for the development of oocytes and early embryos. By analyzing ESTs from a rainbow trout oocyte cDNA library, we identified a novel EST sequence that does not show homology to any sequences in the GenBank. Analysis of tissue distribution by RT-PCR revealed that this gene was only expressed in unfertilized oocytes. Sequencing of the EST clone identified a cDNA of 3163 bp. Northern blot analysis showed the novel gene has a single transcript of 3.4 kb. Additional 5\u27 sequence was obtained by 5\u27 RACE, extending the novel cDNA to 3333 bp. Analysis of the full length cDNA identified an open reading frame encoding a protein of 564 amino acids. The novel protein contains a conserved oxysterol binding protein (OSBP) domain at the C terminus that is characteristic of OSBP-related proteins implicated in lipid metabolism. Therefore, we named the novel gene as Oocyte-specific Oxysterol binding protein Related-Protein of Trout (OORP-T). In situ hybridization showed that the OORP-T mRNA appears to be confined to the cytoplasm of vitellogenic oocytes. Transcription of OORP-T appears to start during pre-vitellogenesis and increases steadily, reaching its peak in the late vitellogenic stage. OORP-T transcript is abundantly present in unfertilized eggs but the level drops significantly in day 2 embryos and continues to decline in day 7 embryos after which it remains low. It is proposed that OORP-T may play an important role in the utilization of yolk derived lipid products during oocyte development and early stages of embryonic development in rainbow trout.;Maternal-zygotic transition (MZT) is the first major transition in early development leading to the activation of embryonic genome. Effective transcription machinery including transcription factors must be in place during MZT for it to occur. Therefore, measuring the transcript abundance of key transcription factors prior to and after MZT can give important clues about the roles of transcription factors during this process. In this study, we quantitatively measured mRNA abundance of 9 selected transcription factors (Figla, P300, YY1, HMGA1, HMGB1, HMGN1, ATF-1, TEAD2 and OCT-4) in unfertilized eggs and early stage embryos from day 1 through day 7 post fertilization using quantitative real time PCR. Our results demonstrate that significant amounts of mRNA for all transcription factors studied are present in unfertilized eggs and day 1 embryos, and the expression of all transcription factors reaches minimum levels in day 2 embryos. While some transcription factors remain at low levels of expression throughout late stage development, others show significant increase of expression following embryonic genome activation. The expression patterns of these transcription factors are suggestive of their roles in MZT as well as in early development in rainbow trout.;Current literature and our results on expression patterns of oocyte specific genes and transcription factors suggest global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). We hypothesized that microRNAs (miRNAs), naturally occurring 19-21bp long post-transcriptional regulators, are involved in this degradation process. We analyzed the expression pattern of dicer, an enzyme required for the processing of microRNAs. Dicer is abundantly expressed until 24 hours post-fertilization and gets down-regulated afterwards. This supports the hypothesis that dicer processes mature miRNAs during these stages and these miRNAs in turn degrade maternal mRNAs. To identify candidate microRNAs involved in this process, we constructed a miRNA library from a pool of oocytes and early stage embryos (0 hour post-fertilization through 96 hours post-fertilization). Sequencing analysis of clones showed that there are at least 15 miRNAs expressed during these stages, 4 of which are novel to rainbow trout. We carried out quantitative real-time PCR to learn more about their expression pattern. Our results show that several microRNAs are up-regulated when maternal RNAs are degraded. Stat3, a transcription factor which is involved in activating the transcription of miR-21 is also abundantly expressed in early rainbow trout embryos. Taken together, these results indicate that up-regulated microRNAs, some induced by stat3, could be responsible for degradation of maternal mRNAs in early embryos.;Identification and characterization of a novel oocyte specific gene with a conserved domain that is involved in oxysterol (a metabolite of cholesterol, a precursor molecule of all steroid hormones) metabolism is described here. Expression pattern of OORP-T follows the pattern of estrogen during oogenesis indicating its unique role in oogenesis and early embryonic development although the functions of OORP-T remain to be discovered. None the less, the OORP-T can potentially be used as a marker in selecting for high growth, better nutritional efficiency, disease resistance etc. Based on the results of studies on expression pattern of transcription factor mRNAs and microRNAs, it appears that microRNAs may be involved in maternal mRNAs degradation before EGA. The microRNAs identified and characterized here might also serve as markers for above mentioned economically important traits especially because microRNA might be regulating several target genes involved in any of the above mentioned phenotypes

A transcriptome approach towards understanding the development of ripening capacity in Bartlett pears (Pyrus communis L.).

The capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in Bartlett pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity

IDENTIFICATION OF A NON-CLASSICAL GLUCOCORTICOID-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE CHICKEN GROWTH HORMONE GENE

Growth hormone (GH) effects growth and contributes to a lean phenotype in broiler chickens. GH secretion by the anterior pituitary begins on embryonic day (e) 14, concomitantly with a rise in adrenal glucocorticoids (GC) or corticosterone (CORT) secretion. CORT treatment of chicken embryonic pituitary (CEP) cells induces GH secretion prematurely. GC induction of the GH gene requires on-going protein synthesis or an intermediary protein, but the gene lacks a classical GC-response element. We hypothesized that a GC-responsive intermediary protein is necessary for the CORT induced increase in GH. Characterization of the upstream region of the gene may help identify such a protein. To this end, a fragment of the GH gene (-1727/+48) was cloned into a luciferase reporter and characterized in e11 CEP cells. CORT treatment increased luciferase activity and mRNA. Inclusion of CHX blocked CORT induction of luciferase mRNA. Through deletion analysis, we found that a GC-responsive region (GCRR) is located at -1045 to -954. By defining the GC-responsive region and cis-acting elements located within, trans-acting proteins involved in GC induction of the GH gene may be identified. The GCRR is CORT-responsive in either orientation, but it is context-dependent. Potential transcription factor motifs in the GCRR include ETS-1 and a degenerate GRE (GREF). Nuclear proteins bound to a GCRR probe in a CORT-regulated manner and unlabeled competitor DNA competed off binding. Mutation of the central portion of the DNA probe resulted in a significant decrease in protein binding. Mutation of the ETS-1 site or GREF site in the -1045/+48 GH construct resulted in ablation of luciferase activity. ETS-1 and GR are associated with the endogenous gene under basal and 1.5 h CORT-treated conditions, while GR recruitment increased after CORT treatment. GC regulation of the GH gene during chicken embryonic development requires cis-acting elements located 1 kb upstream from the transcription start site and includes recruitment of ETS-1 and GR. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development. Characterization of GC regulation of the GH gene during embryonic development enhances our understanding of growth regulation in vertebrates

Overcoming primary and acquired erlotinib resistance with epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase (PI3K) co-inhibition in pancreatic cancer

PI3K/Akt is over-expressed in 50-70% of pancreatic ductal adenocarcinoma (PDAC). The hypothesis of this study is that PI3K and EGFR co-inhibition may be effective in PDAC with upregulated PI3K/Akt/mTOR (PAM) signaling. Five primary PDAC and two erlotinib acquired resistant (ER) cell lines with significantly over-expressed AKT2 gene, total Akt and pAkt, were used. Multiple inhibitors of the MAPK and PAM were tested alone or in combination by western blotting, cell proliferation, cell cycle, clonogenic, apoptosis, and migration assays. Erlotinib acted synergistically with PI3Kα inhibitor BYL in both ER cell lines (synergy index, SI=1.71 and 1.44 respectively). Treatment of ER cell lines by this dual blockade caused significant G1 cell cycle arrest (71%, P<0.001; 58%, P=0.003), inhibition of colony formation (69% and 72%, both P<0.001), and necrosis and apoptosis (75% and 53%, both P<0.001), more so compared to parent cell lines. In primary patient-derived tumor subrenal capsule (n=90) and subcutaneous (n=22) xenografts, Erlotinib plus BYL significantly reduced tumor volume (P=0.005). Strong pEGFR and pAkt immunostaining (2+/3+) was correlated with high response to erlotinib and low response to erlotinib plus BYL respectively. In conclusion, PDAC with increased expression of the PAM signaling were susceptible to PI3K/ EGFR co-inhibition suggesting oncogenic dependence. Erlotinib plus BYL should be considered for a clinical study in PDAC; further evaluation of pEGFR and pAkt expression as potential predictive biomarkers is warranted

Ontogenic and glucocorticoid-regulated gene expression in the developing neuroendocrine system

The neuroendocrine system is a critical regulator of vertebrate homeostasis that includes five hypothalamic-pituitary axes which develop during embryogenesis. Adrenal glucocorticoids play an important role in functional maturation of the anterior pituitary through initiation of growth hormone (GH) production. These studies were aimed at characterizing ontogenic and glucocorticoid-regulated changes in gene expression during neuroendocrine system development in the chick. First, to ascertain timing of initiation and establishment of each neuroendocrine axis, we measured mRNA levels of hypothalamic regulatory factors, their pituitary receptors, and pituitary hormones from embryonic day (e) 10 through post-hatch day (d) 7. We found that the adrenocorticotropic axis is the first to be established (e12), followed by establishment of the thyrotropic (e18), somatotropic (e20), lactotropic (d1), and gonadotropic (d5) axes. Next, we examined in detail mechanisms through which glucocorticoids initiate pituitary GH expression during embryogenesis. We determined that glucocorticoids elevate GH mRNA levels on e11 by increasing transcriptional activity of the GH gene rather than enhancing mRNA stability, and protein synthesis, histone deacetylase activity, ras signaling, and ERK1/2 signaling are required for this activation. Conversely, sustained activation of ERK1/2 and p38MAPK pathways reduced glucocorticoid stimulation of GH expression, indicating the requirement for ERK1/2 activity is transitory. Finally, we identified ras-dva as a novel Pit-1 and glucocorticoid-regulated gene in the chicken embryonic pituitary gland. Pituitary ras-dva mRNA levels increased between e10 and e18, decreased just prior to hatch, and remained low or undetectable post-hatch. Ras-dva expression was highly enriched within the pituitary gland on e18, and glucocorticoids rapidly induced ras-dva mRNA in cultured pituitary cells through a mechanism involving transcriptional activation. Potential regulatory elements within the 5'-flanking region of chicken ras-dva responsible for pituitary-specific expression were identified, as was a 2 kb fragment necessary for its glucocorticoid induction in embryonic pituitary cells. These results enhance our understanding of neuroendocrine system development and establishment during embryogenesis, reveal mechanisms underlying glucocorticoid initiation of GH expression in somatotrophs, and identify a new Pit-1 and glucocorticoid target gene that may play an important role in pituitary development

Tissue specific action of Gibberellin in Arabidopsis flowering and development

This work focuses on understanding the roles of the plant hormone gibberellin (GA) in controlling the initiation of flowering in Arabidopsis thaliana. GA is essential to promote the transition to flowering under non inductive short-­‐day (SD) photoperiods by activating transcription of the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and of the meristem identity gene LEAFY (LFY). However, mutations in GA receptors also prevent flowering under inductive long days (LDs), suggesting that this hormone also has crucial functions in the initiation of flowering under these conditions. Here by overexpressing the GA catabolic enzyme GIBBERELLIN 2 OXIDASE 7 (GA2ox7) in specific plant organs, we show that GAs play important regulatory roles in the leaves and shoot apical meristem (SAM) to promote flowering under LDs. Our results suggest that GAs are required in the leaf to increase levels of FT mRNA, which encodes a protein that is part of the systemic florigen signal of Arabidopsis. At the SAM GAs promote expression of SQUAMOSA PROMOTER BINDING PROMOTER LIKE (SPL) genes downstream of the floral integrator SOC1. In addition, we characterised a novel function of the MADS box transcription factor SHORT VEGETATIVE PHASE (SVP) and demonstrated its link to the GA biosynthetic pathway at the SAM. Mutation of SVP results in a significant accumulation of active GA4 through the upregulation of GIBBERELLIN 20-­‐OXIDASE 2 (GA20ox2), which encodes an enzyme involved in GA biosynthesis. Conversely overexpression of SVP from the 35S promoter causes phenotypes characteristic of GA deficiency plants. We demonstrate that the SVP/GA20ox2 module is controlled by photoperiod through FT, TSF and SOC1 at the SAM. Wild-­‐type plants shifted from SDs to LDs showed downregulation of SVP in the centre of the SAM and increased levels of GA20ox2 transcripts in the rib meristem region. These expression patterns are significantly compromised in plants lacking FT, TSF or SOC1 functions. We propose that in response to LDs, FT, TSF and SOC1 act to repress expression of SVP leading to upregulation of GA20ox2. The activation of GA20ox2 expression causes increased GA content, which promotes flowering by activating transcription of SPL genes. Finally, we identified a link between a core subunit of chromatin remodelling complexes (CRCs) SWI3C and the GA signalling and biosynthesis pathways. The swi3c mutant displayed several developmental impairments, which resembled those of GA deficient plants. In agreement with the phenotypic characterization, swi3c mutants showed lower levels of active GAs and reduced mRNA abundance of the GA receptor GIDa, suggesting that SWI3C is required to control development by modulating GA biosynthesis and perception. Moreover we demonstrate that SWI3C binds in vivo to some of the DELLA repressors (RGA, RGL1, RGL2, RGL3) and SPY O-­‐GlcNAc transferase, two components of the GA signalling pathway. Our results indicate that CRCs control plant development at least in part by promoting GA biosynthesis, and by regulating expression of some GA responsive genes. Overall this work increases our understanding of the regulation of GA biosynthesis and signalling, as well as demonstrating new functions for these processes in the control of the floral transition

Mapping quantitative trait loci associated with chilling requirement and bloom date in peach

Chilling requirement (CR), together with heat requirement (HR), determines bloom date (BD), which impacts climatic distribution of genotypes of temperate tree species. The molecular basis of floral bud CR is poorly understood despite its importance to fruit tree adaptation and production. A peach F2 populations developed from two genotypes with contrasting CR values was used for QTL mapping for CR, HR and BD. Using the Contender × Fla.92-2c population, 20 QTLs with additive effects were identified for three traits including one major QTL for CR and two major QTLs for BD. Particularly, one genomic region of 2cM pleiotropic for the three traits overlaps with the sequenced peach evg region. Association approaches and candidate-gene approaches were used to explore and refine the detected QTL regions. Seven Polycomb group and their associated protein encoding genes in/close to QTL regions and three genes (including DAM4 and DAM6) in evg locus were identified as potential candidate genes regulating CR and BD. In addition, we established the use of plum as transgenic system to test peach candidate genes for CR and BD. Transgenic plums overexpressing DAM6 showed dwarfing and more branching phenotype

Plant defence responses against Radopolus similis in East African Highland bananas (EAHB- AAA) inoculated with endophytic non-pathogenic Fusarium oxysporum

In the interactions between fungal endophytes and their hosts, the host may benefit through protection against pathogens and pests, growth promotion and tolerance to abiotic stresses. Non-pathogenic Fusarium oxysporum endophytes of banana have been shown to reduce the damage caused by the Cosmopolitus sordidus and the burrowing nematode Radopholus similis. The mode of protection against the burrowing nematode involves induced resistance, but the molecular basis of this resistance yet to be demonstrated. It has further been reported that protection of the host by multiple endophytes can lead to better control of target pests, probably because of the multiple modes of action involved. This phenomenon, however, has not been fully demonstrated for F. oxysporum endophytes of banana. This study aimed to investigate the molecular and biochemical basis of endophyte protection of East African Highland bananas (EAHB) against C. sordidus and R. similis. Expression of banana defence-related genes following endophyte inoculation and R. similis challenge varied greatly between the nematode-susceptible cv Nabusa and the nematode-tolerant cv Kayinja. In cv Nabusa, only the peroxidase (POX) and lectin genes were responsive to endophyte colonization of roots, or R. similis challenge. POX and lectin activities were significantly down-regulated 2 and 33 days after endophyte inoculation (dai), respectively. In cv Kayinja, endophyte colonization resulted in transient up-regulation of POX and a down-regulation of endochitinase (PR-3), lectin, pectin acetylesterase (PAE), phenylalanine ammonia-lyase (PAL) and PIR7A (peroxidase). Similar to systemic acquired resistance, PR-1 and catalase activities were up-regulated in the cv Kayinja 33 dai. Genes involved in signal transduction, cell wall strengthening, jasmonic acid pathway and defence molecule transport were differentially expressed in endophyte-inoculated plants. The expression profiles of four defence-related genes following endophyte inoculation and R. similis challenge were studied using quantitative real-time PCR. ABC transporter, Β-1,3-glucan synthase, coronatine insensitive 1 (COI1) and lipoxygenase (LOX) were up-regulated following endophyte inoculation. Β-1,3-glucan synthase and COI1 were highly up-regulated following R. similis challenge of endophyte-inoculated plants of the susceptible cv Nabusa, while COI1 and LOX were highly up-regulated following nematode challenge of endophyte-inoculated plants of the tolerant cv Kayinja. However ABC transporter gene activity was not up-regulated following nematode challenge of plants of both cultivars. UP-regulation of phenylpropanoid pathway enzymes PAL, POX and PPO has been observed in roots following colonization by both pathogenic and non-pathogenic fungi. In the current study, endophyte inoculation resulted in down-regulation of PAL activity in both a susceptible (cv Nabusa) and tolerant (cv Yangambi) banana. In cv Nabusa, endophyte inoculation primed PAL activity for up-regulation 30 days post nematode challenge (dpnc). However, in cv Yangambi PAL activity was up-regulated 7 dpnc irrespective of endophyte inoculation. Endophyte inoculation transiently up-regulated POX in cv Nabusa, but activity reduced to the levels in the controls 30 dai. Similar to PAL, R. similis challenge of endophyte-inoculated plants of Nabusa caused significant up-regulation of POX 7 dpnc. Nematode challenge of control plants of cv Yangambi resulted in a non-significant up-regulation of POX compared with non-challenged controls, but a significant up-regulation compared to all endophyte-inoculated plants. PPO activity was transiently up-regulated in cv Nabusa and down-regulated in cv Yangambi 7 dai. For all treatments, PPO activity was significantly reduced between 7 dai and 120 dai (60 dpnc). Fusarium oxysporum endophyte isolates Emb2.4o and V5w2 were successfully marked with benomyl- and chlorate resistance and transformed with fluorescent protein genes, while Eny1.31i, Eny7.11o and V4w5 were marked with benomyl resistance only. Most mutants and fluorescent protein transformants maintained resistance to the selective chemical on PDA and after plant colonization. Benomyl- and chlorate-resistant mutants were successfully used to determine actual plant colonization percentages by inoculated endophytes. Similarly, GFP transformants were successfully used to ascertain the pattern of endophytic root colonization in vivo. In plants dually inoculated with isolates Emb2.4o BR 8 and V5w2 CHR 9, both isolates were recovered from roots and rhizomes 4 weeks after inoculation, but isolate V5w2 CHR 9 proved a better colonizer of the two tissue types. Root colonization by isolate V5w2 CHR 9 was boosted when inoculated dually with Emb2.4o BR 8, while that by Emb2.4o BR 8 was reduced in the presence of V5w2 CHR 9. Where growth advantages were observed for dually inoculated plants, it occurred where plants were challenged with R. similis. In the absence of pests, control plants showed better growth than endophyte-inoculated plants. On the other hand, weevil challenge of control plants resulted in significant reductions in plant height, number of live roots and root fresh weight. Dual endophyte inoculation resulted in a significant reduction in R. similis populations in nematode only challenged plants, compared with plants inoculated with Emb2.4o BR 8 singly and control plants challenged with the nematode. In one replicate banana weevil damage to the outer and inner pseudostem base, and the inner rhizome were significantly reduced for dually-inoculated plants. CopyrightThesis (PhD)--University of Pretoria, 2008.Microbiology and Plant Pathologyunrestricte

Genetic variation of traits related to salt stress response in Wheat (<em>Triticum aestivum</em> L.)

Salinity is one of the most severe abiotic stresses perceived by plants, and is continuously increasing due to climatic change and poor irrigation management practices. It is currently affecting ~800 million hectares of land worldwide, including over 20% of the world’s irrigated arable land. Salinity causes significant growth reduction and crop yield losses. With the predicted geometric increase in the global population, improving the salt tolerance (ST) of crops has become an important challenge and target for plant breeders. Several approaches have been exhaustively exploited to ameliorate the impact of salinity on crop plants, but because of the complex nature of ST in crop plant, these approaches have not been optimally translated into the desired results. It is well known that ST is difficult to breed due to its interaction with many physiological processes that are controlled by many genes, plant growth stage and are influenced by environmental factors. Wheat is moderately salt tolerant which means that the grain yield is significantly affected under soil saline condition of ~10 dS m-1. Therefore, improving wheat adaptation under high salinity is seen as the most efficient and economical approach to address the salinity problem and increase its grain yield especially in the poor resource wheat producing countries that are prone to soil salinity. This thesis applies several morphological and physiological evaluations, genetic and molecular approaches to elucidate the genetic and physiological mechanisms underlying natural variation for ST in wheat and to find ways to explore the inherent genetic variation, with the ultimate aim of finding new candidate genes that can be used to improve ST in wheat. The performance of 150 genetically diverse wheat genotypes were evaluated under different salinity conditions at germination, seedling and adult plant field growth stages, to identify heritable variation for salt tolerance in the measured traits. In addition, the amount of Na+, K+ and K+/Na+ ratio in the different shoot parts such as third leaves, stem and remaining leaf parts were determined for each genotypes after 24 days of stress under 150 mM/L NaCl. Results revealed genotype and salt treatment effects across all the growth stages, and the salt stress applied caused 33%, 51% and 82% reductions in germination vigour, seedling biomass and grain yield, respectively. The ability of wheat to conserve water in both root and shoot tissues was positively correlated with the K+ uptake under exposure to salinity. The wide-spectrum of responses to salt stress observed among the genotypes was exploited to identify genotypes with most consistent ST status across growth stages. Among the outstanding genotypes identified, four genotypes including Altay2000, 14IWWYTIR-19 and UZ-11CWA-8 (tolerant) and Bobur (sensitive) showed consistent ST status across the three growth stages including germination, seedling and adult-plant field growth stages. Further evaluation of the identified genotypes using several physiological parameters showed that the tolerant genotypes possess better adaptation characteristics than the sensitive ones (Bobur and UZ-11CWA-24) which allowed them to sustain growth and reproduce under high salinity. A high density molecular map with ~18,000 SNPs (average distance between markers of 0.49 cM cM) and all the morpho-physiological and seed quality data collected were used to map QTLs for ST in the studied population. The LD decayed moderately fast (10 cM, 11 cM and 14 cM (r2 > 0.1) for the A, B and D-genome, respectively). By applying mixed linear modeling (MLM) while correcting for the effects of population structure and the kinship resulted in the detection of 302 SNPs (representing 50 distinct QTL regions) that were significantly associated with various ST traits. They explained between 2.00 and 63.45 % of the genetic variance. Most of the associated SNPs/loci showed pleiotropic effect on several traits and/or were detected across several independent experiments/growth stages. For instance, a single locus (at 90.04 cM) on 6AL was found to be strongly associated with ABS/RC, DIo/RC and shoot Na+ traits. An important (about 1.8 cM interval) region on 2BL was also found to strongly contribute to the variation in ST in various salt stress related traits (ST_DRW, shoot Na+, Fv/Fm, grain yield and seed crude protein). Five novel ST QTL regions were also detected on 1BS, 1DL, 5BS, 6AL and 5BL genomic regions. All the identified QTL have been discussed in this thesis. By analyzing sequences of the associated SNPs, several key genes involved in salt and abiotic stress tolerance were identified. Among the categories of genes identified (Chapter 3 and 4), the genes involved in the stress response (24%), antiporter and transmembrane (18%), transcription and translation (14%), and redox homeostasis and detoxification (11%) related activities occurred predominantly. The transcriptome and RT-PCR expression analyses performed with the genes linked to the significant MTAs revealed differential expressions between the contrasting ST wheat genotypes. Moreover, the amino acid sequence analyses of the putative genes uncovered many sites of non-synonymous/missense mutation that may have contributed to the observed variable salt stress responses in the contrasting wheat genotypes. This study provides new insights towards understanding the traits and mechanisms related to ST. Thus, the underlying genetic and molecular response as presented in this thesis can be directly exploited by the breeders and scientists to improve salt tolerance in wheat.Die Versalzung des Bodens zählt zu den größten abiotischen Stressfaktoren für Pflanzen, und steigt durch den Klimawandel und ein schlechtes Wassermanagement kontinuierlich. Zur Zeit sind etwa 800 Millionen Hektar weltweit und 20 % der künstlich bewässerten Flächen von Versalzung betroffen. Diese führt zu einer signifikanten Reduktion des Pflanzenwachstums und ist mitverantwortlich für Ertragseinbußen. Durch das weltweite Bevölkerungswachstum wird die Erhöhung der Salztoleranz (ST) von Nutzpflanzen eine immer wichtigere Aufgabe und ein anzustrebendes Ziel für die Pflanzenzüchtung. Verschiedene Forschungsansätze wurden verfolgt, um die Salztoleranz von Pflanzen zu verbessern, jedoch führten viele dieser Ansätze aufgrund der komplexen Natur der ST nicht zu verwertbaren Ergebnissen. Es ist bekannt, dass ST aufgrund der Interaktion zwischen vielen physiologischen Prozessen, den unterschiedliche Genen und der Umwelt, schwierig in die Züchtung zu integrieren ist. Weizen gilt als mäßig salztolerant und der Ertrag wird ab einem Bodensalzgehalt von ~10 dS m-1 signifikant beeinflusst. Gerade die landwirtschaftlich schwächer entwickelten Regionen sind für Bodenversalzung anfällig und eine Erhöhung der Salztoleranz wäre ein probates wirtschaftliches Mittel um den Weizenertrag zu steigern. Diese Dissertation nutzt mehrere morphologische und physiologische Auswertungen, genetische und molekulare Ansätze, um die genetischen und physiologischen Mechanismen zu erklären, die der ST des Weizens zugrunde liegen. Dabei soll die eigene genetische Variation des Weizens erklärt und schlussendlich neue Kandidatengene gefunden werden, welche die ST des Kulturweizens erhöhen. Die Leistung von 150 genetisch verschiedenen Weizengenotypen wurde während der Keimung, dem Sämlingsstadium und an der adulten Pflanze unter unterschiedlichen Salzbedingungen geprüft, um die erbliche Variation des ST in unterschiedlichen Merkmalen oder Wachstumsstadien zu identifizieren. Nach 24 Stunden unter Stressbedingungen mit 150 mM/L NaCl wurde der Na+-, K+- Gehalt und des K+/Na+ - Verhältnis in verschiedenen Sprossteilen, wie dem dritten Blatt, dem Stängel und den übrigen Blättern für alle Genotypen bestimmt. Die Ergebnisse zeigten Interaktionen der Genotypen und der Salzbehandlung in allen Wachstumsstadien. Die Salzapplikation verursachte einen Rückgang von 33% bei der Keimfähigkeit, von 51 % der Sämlingsbiomasse und von 82% beim Kornertrag. Die Eigenschaft des Weizens, Wasser in Wurzel- und Sprossteilen zu speichern war positiv mit der K+ -Aufnahme unter Stressbedingungen korreliert. Das beobachtete breite Spektrum der Pflanzenreaktionen auf die Salzstressapplikation wurde genutzt um die beständigsten, beziehungsweise die salztolerantesten Genotypen über alle Wachstumsstadien zu identifizieren. Es wurden vier extreme Genotypen (Altay2000, 14IWWYTIR-19 und UZ-11CWA-8 (tolerant) und Bobur (sensitiv)) ausgewählt, die eine konstante ST über die untersuchten Wachstumsstadien zeigten. Weitere Tests der ausgewählten Genotypen mit verschiedenen physiologischen Parametern zeigten, dass die toleranten Genotypen über bessere Anpassungsmechanismen verfügen als die sensitiven (Bobur und UZ-11CWA-24). Dadurch ist es ihnen möglich, auch unter hohem Salzgehalt das Wachstum aufrecht zu erhalten und fertil zu bleiben. Eine hochauflösende molekulare Karte mit ~18000 SNPs und einer durchschnittlichen Distanz zwischen den Markern von 0.49 cM wurde zusammen mit den gesammelten morphologischen-, physiologischen- und Saatgutqualitätsdaten genutzt um QTLs für die ST der untersuchten Population zu bestimmen. Das LD der Weizenpopulation liegt bei 10 cM auf dem A-, bei 11 cM auf dem B- und bei 14 cM auf dem D-Genom bei einem r² > 0.1. Mittels gemischten linearen Modellen (MLM) und deren Korrektur durch die Verwandtschaftsmatrix, wie auch die Populationsstruktur wurden 302 SNPs in 50 verschiedenen QTL Regionen detektiert, die signifikant mit verschiedenen Merkmalen für ST assoziiert sind. Diese SNPs erklären zwischen 2.00 und 63.45 % der genetischen Varianz in der Population. Die meisten assoziierten SNPs/Genorte zeigen pleiotrope Effekte mit mehreren Merkmalen und wurden außerdem in unabhängigen Experimenten und Wachstumsstadien nachgewiesen. Ein einziger Lokus bei 90.04 cM auf 6AL zeigte zum Beispiel eine starke Assoziation mit den Merkmalen: ABS/RC, DIo/RC und Spross Na+. Eine weitere hervorzuhebende Region mit der Länge von 1.8 cM auf 2BL hatte eine starke Wirkung auf die Variation der ST in den Merkmalen: ST_DRW, Spross Na+, Fv/Fm, Kornertrag und Rohproteingehalt im Samen. Weitere fünf neue ST-QTL Regionen auf 1BS, 1DL, 5BS, 6AL und 5BL wurden gefunden und in dieser Dissertation diskutiert. Durch die Sequenzanalyse assoziierter SNPs wurden mehrere Schlüsselgene identifiziert, welche die Salz- und abiotische Stresstoleranz beeinflussen. Bei den Kategorien der identifizierten Gene (in den Kapiteln 3 und 4) handelt es sich um Gene die mit der: Stressantwort (24%), Antiporter und Transmembran (18%), Transkription und Translation (14%) und redox-gleichgewicht und Entgiftung (11%) verknüpft sind. Transkriptom und RT-PCR-Expressionsanalysen der Marker-Merkmal-Assoziierten (MTA) Gene zeigten, dass diese Gene in den unterschiedliche ST Weizengenotypen unterschiedlich exprimiert wurden. Darüber hinaus wurde die Aminosäuresequenz von einigen Genen überprüft, die wahrscheinlich zu den Salzstressreaktionen beitragen. Diese Studie zeigt neue Einsichten, die zum Verständnis der Merkmale und Mechanismen, die mit ST verbunden sind beitragen. In dieser Dissertation werden genetische und molekulare Ergebnisse präsentiert, die direkt von Züchtern und Wissenschaftlern genutzt werden können, um die Salztoleranz in Weizen zu erhöhen