eCALIBRATOR : a comparative tool to identify key genes and pathways for eucalyptus defense against biotic stressors

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under long-term nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic

The Eucalyptus grandis NBS-LRR gene family : physical clustering and expression hotspots

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.Table S1 Full list of Eucalyptus grandis putative NBS-LRR genes sorted by position on the genome. Information per gene includes the chromosomal position, class, physical cluster and phylogeny clade membership, identification method, raw expression data, log2 fold change values and ANOVA results (p-values). S_F_C, susceptible, fungal treatment, control; S_F_I, susceptible, fungal treatment, inoculated; R_F_C, resistant, fungal treatment, control; R_F_I, resistant, fungal treatment, inoculated; S_I_C, susceptible, insect treatment, control; S_I_I, susceptible, insect treatment, infested; R_I_C, resistant, insect treatment, control; R_I_I, resistant, insect treatment, infested.Table S2 Conserved amino acid sequences for NB-ARC and TIR motifs from MEME analysis with CNL-like and TNL-like gene models in Eucalyptus grandis (Eg) and Arabidopsis thaliana (At; Meyers et al., 2003). The expected amino acid tryptophan (W) is identified in the Kinase 2 subdomain for CNL sequences–underlined.Figure S1 Neighbor joining tree of 480 Eucalyptus grandis NB-ARC domains from complete NBS-LRR genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 495 amino acid sequences (480 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S2 Neighbor joining tree of 616 Eucalyptus grandis NB-ARC domains from all non-TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 631 amino acid sequences (616 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S3 Neighbor joining tree of 396 Eucalyptus grandis NB-ARC domains from all TIR NBS-LRR-like genes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis involved 411 amino acid sequences (396 E. grandis). All ambiguous positions were removed for each sequence pair.Figure S4 The definition of a (A) cluster and a (B) supercluster is illustrated using a region (starting at 13 Mb and ending at 18 Mb) on chromosome 4.Figure S5 Physical locations for all complete, partial, and incomplete NBS-LRR gene models that were expressed under challenge of Chrysoporthe austroafricana and Leptocybe invasa on Eucalyptus grandis chromosomes (Mapchart). Variation in means from treatment (ANOVA) were identified based on significance *p < 0.01, **p < 0.001, ***p < 0.0001 (*** are also underlined) and log2 gene expression ratios greater than 1 or smaller than −1 for resistant and susceptible plants. Color distinguishes between different classes (TNL = pink, CNL = green, NL = red, incomplete NL = black, BLAST homolog non-NL = black). Scale bar = Mb. Cluster and supercluster regions are indicated and E. grandis gene IDs are provided.Figure S6 NB-ARC-LRR fused domains (A) and TIR-NB-ARC-LRR fused domains (B). Conserved amino acid sequences are indicated with lines (top). The GKT (Kinase 1) conserved motif is recognized as a P-loop structure important in ATP hydrolysis while the hDD is also well conserved in NB-ARC domains (Kinase 2) as important in co-ordinating Mg2+ as a co-factor (Tameling et al., 2006). These two important sub-domains of NB-ARC are sometimes termed the Walker A and Walker B motifs (Walker et al., 1982) and are identified as A and B, respectively, within the I-Tasser protein structures (bottom) for a representative CNL (Eucgr.L01363) and TNL (Eucgr.C00020) sequence from the Eucalyptus grandis genome.Top up scholarships were generously provided for PT from the University of Sydney and Rural Industries Research and Development Corporation, Australiahttp://www.frontiersin.orgam2016Genetic

eCALIBRATOR : a comparative tool to identify key genes and pathways for Eucalyptus defense against biotic stressors

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under longterm nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5.Supplementary Material: Figure S1 : Selection of atnrt2.5 T-DNA mutants. (A) Diagram of the AtNRT2.5 gene consisting of three exons and two introns with the positions of the T-DNA insertions in GK213H10 (AtNRT2.5-A) and GK046H04 (AtNRT2.5-B) in the second and first exons, respectively. (B,C) PCR detection of T-DNA in AtNRT2.5-A (B) and AtNRT2.5-B (C), respectively, using a combination of the T-DNA left border oligonucleotide and gene-specific oligonucleotides. No amplification was observed in AtNRT2.5-A and AtNRT2.5-B using gene-specific oligonucleotides spanning the T-DNA insertion sites.Table S1 : Summary of the mapping of RNA-seq libraries per Eucalyptus–pathogen interaction sample.Table S2 : Functionally enriched terms of the gene ontology biological processes category in common between the resistant and susceptible defense responses.Table S3 : Functionally enriched terms of the gene ontology biological processes category in the unique set of differentially expressed genes between the resistant and susceptible interactions.The Department of Science and Technology grant for Forest Genomics and Biotechnology, the South African National Research Foundation Grant for Y-rated researchers (UID105767) Incentive funding for rated researchers (UID95807), Technology and Human Resources for Industry Program (THRIP, Grant ID 96413) and the Technology Innovation Agency (TIA) Forest Molecular Genetics Cluster Program.http://www.frontiersin.org/Microbiologyam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Benefits of maize resistance breeding and chemical control against northern leaf blight in smallholder farms in South Africa

Maize underpins food security in South Africa. An annual production of more than 10 million tons is a combination of the output of large-scale commercial farms plus an estimated 250 000 ha cultivated by smallholder farmers. Maize leaves are a rich source of nutrients for fungal pathogens. Farmers must limit leaf blighting by fungi to prevent sugars captured by photosynthesis being ‘stolen’ instead of filling the grain. This study aimed to fill the knowledge gap on the prevalence and impact of fungal foliar diseases in local smallholder maize fields. A survey with 1124 plant observations from diverse maize hybrids was conducted over three seasons from 2015 to 2017 in five farming communities in KwaZulu-Natal Province (Hlanganani, Ntabamhlophe, KwaNxamalala) and Eastern Cape Province (Bizana, Tabankulu). Northern leaf blight (NLB), common rust, Phaeosphaeria leaf spot, and grey leaf spot had overall disease incidences of 75%, 77%, 68% and 56%, respectively, indicating high disease pressure in smallholder farming environments. NLB had the highest disease severity (LSD test, p<0.05). A yield trial focused on NLB in KwaZulu-Natal showed that this disease reduced yields in the three most susceptible maize hybrids by 36%, 71% and 72%, respectively. Eighteen other hybrids in this trial did not show significant yield reductions due to NLB, which illustrates the progress made by local maize breeders in disease resistance breeding. This work highlights the risk to smallholder farmers of planting disease-susceptible varieties, and makes recommendations on how to exploit the advances of hybrid maize disease resistance breeding to develop farmer-preferred varieties for smallholder production. SIGNIFICANCE : • Northern leaf blight, grey leaf spot, Phaeosphaeria leaf spot and common rust diseases were widespread in KwaZulu-Natal and Eastern Cape smallholder maize fields where fungicides were not applied. • NLB was the most severe maize leaf disease overall. • NLB caused maize leaf blighting, which reduced grain yields by 36–72% in susceptible maize hybrids. • Maize resistance breeding has produced locally adapted hybrids that do not have significant yield losses under NLB disease pressure.Department of Agriculture, Forestry and Fisheries Research Technology Fund through the National Research Foundation of South Africa; USAID through the University of California Davis Research and Innovation Fellowship for Agriculture.http://www.sajs.co.zaam2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution

A genome-wide SNP genotyping resource for tropical pine tree species

We performed gene and genome targeted SNP discovery towards the development of a genome-wide, multispecies genotyping array for tropical pines. Pooled RNA-seq data from shoots of seedlings from five tropical pine species was used to identify transcript-based SNPs resulting in 1.3 million candidate Affymetrix SNP probe sets. In addition, we used a custom 40 K probe set to perform capture-seq in pooled DNA from 81 provenances representing the natural ranges of six tropical pine species in Mexico and Central America resulting in 563 K candidate SNP probe sets. Altogether, 300 K RNA-seq (72%) and 120 K capture-seq (28%) derived SNP probe sets were tiled on a 420 K screening array that was used to genotype 576 trees representing the 81 provenances and commercial breeding material. Based on the screening array results, 50 K SNPs were selected for commercial SNP array production including 20 K polymorphic SNPs for P. patula, P. tecunumanii, P. oocarpa and P. caribaea, 15 K for P. greggii and P. maximinoi, 13 K for P. elliottii and 8K for P. pseudostrobus. We included 9.7 K ancestry informative SNPs that will be valuable for species and hybrid discrimination. Of the 50 K SNP markers, 25% are polymorphic in only one species, while 75% are shared by two or more species. The Pitro50K SNP chip will be useful for population genomics and molecular breeding in this group of pine species that, together with their hybrids, represent the majority of fast-growing tropical and subtropical pine plantations globally.DATA AVAILABILITY STATEMENT : The pooled targeted capture sequencing data have been made available via NCBI SRA BioProject accession PRJNA742386. RNA-seq data are available via NCBI SRA BioProject accessions PRJNA416697 (P. tecunumanii), PRJNA416698 (P. patula), PRJNA685280 (P. oocarpa), PRJNA685281 (P. greggii) and PRJNA685282 (P. maximinoi). Metadata and probe set sequences used for markers selected for the 50 K commercial array are available as Supporting Information (Table S5). Genotype data set used for PCA and STRUCTURE analysis is available in Supporting Information (Table S6).http://www.wileyonlinelibrary.com/journal/men2022-08-12hj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Causal effect of plasminogen activator inhibitor type 1 on coronary heart disease

Background--Plasminogen activator inhibitor type 1 (PAI-1) plays an essential role in the fibrinolysis system and thrombosis. Population studies have reported that blood PAI-1 levels are associated with increased risk of coronary heart disease (CHD). However, it is unclear whether the association reflects a causal influence of PAI-1 on CHD risk. Methods and Results--To evaluate the association between PAI-1 and CHD, we applied a 3-step strategy. First, we investigated the observational association between PAI-1 and CHD incidence using a systematic review based on a literature search for PAI-1 and CHD studies. Second, we explored the causal association between PAI-1 and CHD using a Mendelian randomization approach using summary statistics from large genome-wide association studies. Finally, we explored the causal effect of PAI-1 on cardiovascular risk factors including metabolic and subclinical atherosclerosis measures. In the systematic meta-analysis, the highest quantile of blood PAI-1 level was associated with higher CHD risk comparing with the lowest quantile (odds ratio=2.17; 95% CI: 1.53, 3.07) in an age- and sex-adjusted model. The effect size was reduced in studies using a multivariable-adjusted model (odds ratio=1.46; 95% CI: 1.13, 1.88). The Mendelian randomization analyses suggested a causal effect of increased PAI-1 level on CHD risk (odds ratio=1.22 per unit increase of log-transformed PAI-1; 95% CI: 1.01, 1.47). In addition, we also detected a causal effect of PAI-1 on elevating blood glucose and high-density lipoprotein cholesterol. Conclusions--Our study indicates a causal effect of elevated PAI-1 level on CHD risk, which may be mediated by glucose dysfunction

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Transcriptional regulation underlying the quantitative genetic response of maize to grey leaf spot disease

Cercospora zeina causes grey leaf spot (GLS), a yield-limiting disease on maize. The main objective of this study was to exploit maize gene expression data to dissect the quantitative disease response to C. zeina infection. The project addresses the hypothesis that there is an underlying DNA polymorphism that gives rise to a change in gene expression, which in turn affects GLS disease severity. Genomic and functional annotation of the reporters on an Agilent 44K maize microarray was carried out. This microarray was used for global gene expression profiling of earleaf samples collected from 100 recombinant inbred sub-tropical maize lines exposed in the field to C. zeina. Gene expression profiles together with GLS severity scores were used in a weighted gene co-expression network analysis to identify co-expression modules associated with disease severity. Quantitative trait locus (QTL) mapping for GLS severity was combined with expression QTL (eQTL) analyses to investigate the molecular basis of the quantitative response to GLS. An Eqtl data analysis pipeline was developed in Galaxy. The overlap of phenotypic QTLs with cis- and trans-eQTLs revealed putative causal candidate genes and potential mechanisms responsible for the QTLs, respectively. Regulatory network models were constructed for trans-eQTL hotspots coinciding with phenotypic QTLs. A genetic basis for coordinated expression responses to GLS disease was identified. For the susceptible response, the results lead to the hypothesis that a calmodulin-related protein with a cis-eQTL acts as a global regulator of various pathogenesis-related proteins that are activated too late after infection started. For the resistant response, it is hypothesised that a serine threonineprotein kinase with a cis-eQTL acts as a post-translational global regulator regulating phosphatases and kinases involved in activation of defense gene expression. The outcomes of this study were: i) the development of a systems genetics strategy and ii) several hypotheses of maize transcriptional responses to C. zeina which need to be validated with further studies. These results extend the current knowledge of GLS resistance and could aid in the improvement of maize varieties.Thesis (PhD)--University of Pretoria, 2014.BiochemistryPhDUnrestricte