Improvements to the maize (Zea mays L.) in vivo maternal doubled haploid system

The maize (Zea mays L.) in vivo maternal doubled haploid system has been widely applied to maize breeding and genetics in recent decades and is an important part of the majority of public and private maize breeding programs today. The principal advantage of the doubled haploid system is the ability to generate completely homozygous inbred lines in as little as two seasons. Other advantages to this system include more rapid integration of loci of interest and increased usefulness over traditional lines developed through self-pollination. In this dissertation, some of the major problems in the maternal doubled haploid system are addressed. Namely, improvement of maternal inducers, improved understanding of the genetics controlling inducibility, development of an automated system to sort haploid kernels, investigation and application of spontaneous chromosome doubling, and a proposal for the acceleration of the breeding cycle beyond doubled haploids through the in vitro nursery. This dissertation provides some new insight into these problems, as follows. The development and release of a new improved maternal haploid inducer for use in doubled haploid programs. Improved understanding of the quantitative nature of inducibility and the effects of misclassification are discussed. Successful automated discrimination of haploid and diploid kernels using optical and fluorescence methods is described. In an effort to make the doubled haploid system more efficient and safe, a bypass of the colchicine doubling step is proposed through the application and investigation of spontaneous chromosome doubling in haploid plants. Finally, as a proposal for what could be the next step in accelerating the breeding cycle, the in vitro nursery and its applications is discussed

Breeding Maize for Drought Tolerance: Diversity Characterization and Linkage Disequilibrium of Maize Paralogs ZmLOX4 and ZmLOX5

Maize production is limited agronomically by the availability of water and nutrients during the growing season. Of these two limiting factors, water availability is predicted to increase in importance as climate change and the growing urban landscape continue to stress limited supplies of freshwater. Historically, efforts to breed maize for water-limited environments have been extensive; especially in the areas of root architecture and flowering physiology. As progress has been made and new traits have been discovered and selected for, the different responses to drought stress at specific developmental stages of the maize plant have been selected as a whole when drought tolerance is evaluated. Herein we attempt to define the characteristics of the maize drought response during different developmental stages of the maize plant that can be altered through plant breeding. Towards breeding for drought tolerance, 400 inbred lines from a diversity panel were amplified and sequenced at the ZmLOX4 and ZmLOX5 loci in an effort to characterize their linkage disequilibrium and genetic diversity. Understanding these characteristics is essential for an association mapping study that accompanies this project, searching for novel and natural allelic diversity to improve drought tolerance and aflatoxin resistance in maize. This study is among the first to investigate genetic diversity at important gene paralogs ZmLOX4 and ZmLOX5 believed to be highly conserved among all Eukaryotes. We show very little genetic diversity and very low linkage disequilibrium in these genes, but also identified one natural variant line with knocked out ZmLOX5, a variant line missing ZmLOX5, and five line variants with a duplication of ZmLOX5. Tajima's D test suggests that both ZmLOX4 and ZmLOX5 have both been under neutral selection. Further investigation of haplotype data revealed that ZmLOX12, a member of the ZmLOX family, showed strong LD that extends much further than expected in maize. Linkage disequilibrium patterns at these loci of interest are crucial to quantify for future candidate gene association mapping studies. Knockout and copy number variants of ZmLOX5, while not a surprising find, are under further investigation for crop improvement

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Expenditure Reform in Industrialised Countries: A Case Study Approach

This study examines reforms of public expenditure in industrialised countries over the past two decades. We distinguish ambitious and timid reformers and analyse in detail reform experiences in eight case studies of ambitious reform episodes. We find that ambitious reform countries reduce spending on transfers, subsidies and public consumption while largely sparing education spending. Such expenditure retrenchment is also typically part of a comprehensive reform package that includes improvements in fiscal institutions as well as structural and other macroeconomic reforms. The study finds that ambitious expenditure retrenchment and reform coincides with large improvements in fiscal and economic growth indicators

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Improvements to the maize (Zea mays L.) in vivo maternal doubled haploid system

The maize (Zea mays L.) in vivo maternal doubled haploid system has been widely applied to maize breeding and genetics in recent decades and is an important part of the majority of public and private maize breeding programs today. The principal advantage of the doubled haploid system is the ability to generate completely homozygous inbred lines in as little as two seasons. Other advantages to this system include more rapid integration of loci of interest and increased usefulness over traditional lines developed through self-pollination. In this dissertation, some of the major problems in the maternal doubled haploid system are addressed. Namely, improvement of maternal inducers, improved understanding of the genetics controlling inducibility, development of an automated system to sort haploid kernels, investigation and application of spontaneous chromosome doubling, and a proposal for the acceleration of the breeding cycle beyond doubled haploids through the in vitro nursery. This dissertation provides some new insight into these problems, as follows. The development and release of a new improved maternal haploid inducer for use in doubled haploid programs. Improved understanding of the quantitative nature of inducibility and the effects of misclassification are discussed. Successful automated discrimination of haploid and diploid kernels using optical and fluorescence methods is described. In an effort to make the doubled haploid system more efficient and safe, a bypass of the colchicine doubling step is proposed through the application and investigation of spontaneous chromosome doubling in haploid plants. Finally, as a proposal for what could be the next step in accelerating the breeding cycle, the in vitro nursery and its applications is discussed.</p

Accelerating plant breeding

The growing demand for food with limited arable land available necessitates that the yield of major food crops continues to increase over time. Advances in marker technology, predictive statistics, and breeding methodology have allowed for continued increases in crop performance through genetic improvement. However, one major bottleneck is the generation time of plants, which is biologically limited and has not been improved since the introduction of doubled haploid technology. In this opinion article, we propose to implement in vitro nurseries, which could substantially shorten generation time through rapid cycles of meiosis and mitosis. This could prove a useful tool for speeding up future breeding programs with the aim of sustainable food production.This is a manuscript of an article published as Gerald N.De La Fuente, Ursula K. Frei, and Thomas Lübberstedt. "Accelerating plant breeding." Trends in plant science 18, no. 12 (2013): 667-672. doi: 10.1016/j.tplants.2013.09.001. Posted with permission.</p