Microarray experiments to estimate heterosis

The genetic causes for heterosis, i.e., the increased performance of a hybrid plant compared to the parental mean, may be assessed via microarrays. This thesis addresses design and analysis issues of cDNA-microarray experiments with regard to the estimation of heterosis. Standard microarray designs like the loop design or common reference design are not optimal when estimating heterosis. An optimality criterion is devised and two approaches to obtain a suitable design are shown: a rather intuitive one and an approach using simulated annealing. Data transformations are crucial before analysing microarray data. However, transformations may conceal interesting expression patterns. It is shown using a Box-Cox transformation that significance of a heterotic effect is largely influenced by the transformation parameter. Transformation of the linear predictor in a generalized linear model has a similar effect and heterotic effects may—at least partially—be removed by the transformation. For the estimation of linear contrasts between genotypes, a linear mixed model for each gene is fitted to the expression values. To improve variance estimates one may benefit from other genes’ information. Therefore, an empirical Bayes approach is developed that is capable of including more than one variance component in the model

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

<p>Abstract</p> <p>Background</p> <p>The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, <it>Scmv1 </it>and <it>Scmv2</it>, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.</p> <p>Results</p> <p>By employing time course microarray experiments we identified 68 significantly differentially expressed sequences within the different time points. The majority of differentially expressed genes differed between the near-isogenic line carrying <it>Scmv1 </it>resistance locus at chromosome 6 and the other isogenic lines. Most differentially expressed genes in the SCMV experiment (75%) were identified one hour after virus inoculation, and about one quarter at multiple time points. Furthermore, most of the identified mapped genes were localised outside the <it>Scmv </it>QTL regions. Annotation revealed differential expression of promising pathogenesis-related candidate genes, validated by qRT-PCR, coding for metallothionein-like protein, S-adenosylmethionine synthetase, germin-like protein or 26S ribosomal RNA.</p> <p>Conclusion</p> <p>Our study identified putative candidate genes and gene expression patterns related to resistance to SCMV. Moreover, our findings support the effectiveness and reliability of the combination of different expression profiling approaches for the identification and validation of candidate genes. Genes identified in this study represent possible future targets for manipulation of SCMV resistance in maize.</p

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

Background The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions. Results By employing time course microarray experiments we identified 68 significantly differentially expressed sequences within the different time points. The majority of differentially expressed genes differed between the near-isogenic line carrying Scmv1 resistance locus at chromosome 6 and the other isogenic lines. Most differentially expressed genes in the SCMV experiment (75%) were identified one hour after virus inoculation, and about one quarter at multiple time points. Furthermore, most of the identified mapped genes were localised outside the Scmv QTL regions. Annotation revealed differential expression of promising pathogenesis-related candidate genes, validated by qRT-PCR, coding for metallothionein-like protein, S-adenosylmethionine synthetase, germin-like protein or 26S ribosomal RNA. Conclusion Our study identified putative candidate genes and gene expression patterns related to resistance to SCMV. Moreover, our findings support the effectiveness and reliability of the combination of different expression profiling approaches for the identification and validation of candidate genes. Genes identified in this study represent possible future targets for manipulation of SCMV resistance in maize.This article is published as Użarowska, Anna, Giuseppe Dionisio, Barbara Sarholz, Hans-Peter Piepho, Mingliang Xu, Christina Rønn Ingvardsen, Gerhard Wenzel, and Thomas Lübberstedt. "Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling." BMC Plant Biology 9, no. 1 (2009): 15. doi: 10.1186/1471-2229-9-15. Posted with permission.</p

Heterosis in early seed development: a comparative study of F1 embryo and endosperm tissues 6 days after fertilization

Heterosis specifies the superior performance of heterozygous individuals and although used in plant breeding the underlying molecular mechanisms still remain largely elusive. In this study, we demonstrate the manifestation of heterosis in hybrid maize embryo and endosperm tissue 6 days after fertilization in crosses of several inbred lines. We provide a comparative analysis of heterosis-associated gene expression in these tissues by a combined approach of suppression subtractive hybridization and microarray hybridizations. Non-additive expression pattern indicated a trans-regulatory mechanism to act early after fertilization in hybrid embryo and endosperm although the majority of genes showed mid-parental expression levels in embryo and dosage dependent expression levels in endosperm. The consistent expression pattern within both tissues and both inbred line genotype combinations of genes coding for chromatin related proteins pointed to heterosis-related epigenetic processes. These and genes involved in other biological processes, identified in this study, might provide entry points for the investigation of regulatory networks associated with the specification of heterosis

A first-in-man phase 1 study of the DNA-dependent protein kinase inhibitor peposertib (formerly M3814) in patients with advanced solid tumours

Background: This open-label, phase 1 trial (NCT02316197) aimed to determine the maximum-tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of peposertib (formerly M3814), a DNA-dependent protein kinase (DNA-PK) inhibitor in patients with advanced solid tumours. Secondary/exploratory objectives included safety/tolerability, pharmacokinetic/pharmacodynamic profiles and clinical activity. Methods: Adult patients with advanced solid tumours received peposertib 100–200 mg once daily or 150–400 mg twice daily (BID) in 21-day cycles. Results: Thirty-one patients were included (median age 66 years, 61% male). One dose-limiting toxicity, consisting of mainly gastrointestinal, non-serious adverse events (AEs) and long recovery duration, was reported at 300 mg BID. The most common peposertib-related AEs were nausea, vomiting, fatigue and pyrexia. The most common peposertib-related Grade 3 AEs were maculopapular rash and nausea. Peposertib was quickly absorbed systemically (median Tmax 1.1–2.5 h). The p-DNA-PK/t-DNA-PK ratio decreased consistently in peripheral blood mononuclear cells 3–6 h after doses ≥100 mg. The best overall response was stable disease (12 patients), lasting for ≥12 weeks in seven patients. Conclusions: Peposertib was well-tolerated and demonstrated modest efficacy in unselected tumours. The MTD was not reached; the RP2D was declared as 400 mg BID. Further studies, mainly with peposertib/chemo-radiation, are ongoing. Clinical trial registration: NCT02316197SCOPUS: ar.jinfo:eu-repo/semantics/publishe