Improve small RNA-mediated gene silencing in soybean by using GmFAD3 as a test model

"July 2014."Dissertation Supervisor: Dr. Zhanyuan Zhang.Includes vita.The primary goal of this work is to improve RNAi technology as a tool to analyze gene function and manipulate commercial traits in soybean. We have developed two RNAi approaches towards the silencing of soybean genes: one involved the creation of silenced lines that result from hpRNA-producing transgene, and the second emphasized on the use of an atasiRNA expression cassette. In the first approach, all three family members of GmFAD3 were successfully silenced and the silencing phenotype was stably inherited. Silencing levels of FAD3A, FAD3B and FAD3C correlate to degrees of sequence homology between the inverted repeats (IR) of hpRNA and GmFAD3 transcripts in the RNAi lines. siRNAs generated from the 318-bp IR were characterized and associated with the inferred cleavage sites on target transcripts. Small RNAs corresponding to the loop portion of the hairpin transcript were detected, implicating possible transitive self-silencing of the hairpin transgene. In contrast, much less RNAs were found outside of the target region, suggesting that transitivity along endogenous transcripts is prohibited by some inherent protective feature. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs encoding part of the transgene promoter as well as the bar gene coding sequences were also detected by deep sequencing, but whether they induced the methylation of transgenes still need further exploration. In the second approach, we developed two Arabidopsis TAS1a-based atasiRNA constructs targeting the GmFAD3 gene family using online siRNA design tool OligoWalk. However, computational predicted siRNAs does not represent their in vivo efficacy. Further investigation is needed to determine whether siRNA candidates could conduct efficient silencing of target genes in plants. Furthermore, to simplify the deployment of atasiRNA platform and investigate the utility of miR390 and TAS3 as a gene silencing tool in soybean, spacial and temporal analysIncludes bibliographical references

FFTPL: An Analytic Placement Algorithm Using Fast Fourier Transform for Density Equalization

We propose a flat nonlinear placement algorithm FFTPL using fast Fourier transform for density equalization. The placement instance is modeled as an electrostatic system with the analogy of density cost to the potential energy. A well-defined Poisson's equation is proposed for gradient and cost computation. Our placer outperforms state-of-the-art placers with better solution quality and efficiency

Robust Sensor Networks in Homes via Reactive Channel Hopping

Home area networks (HANs) consisting of wireless sensors have emerged as the enabling technology for important applications such as smart energy and assisted living. A key challenge faced in deploying robust wireless sensor networks (WSNs) for home automation applications is the need to provide long-term, reliable operation in the face of the varied sources of interference found in typical residential settings. To better understand the channel dynamics in these environments, we performed an in-depth empirical study of the performance of HANs in ten real-life apartments. Our empirical study leads to several key insights into designing robust HANs for residential environments. For example, we discover that there is not always a persistently good channel over 24 hours in many apartments; that reliability is strongly correlated across adjacent channels; and that interference does not exhibit cyclic behavior at daily or weekly timescales. Nevertheless, reliability can be maintained through a small number of channel hops. Based on these insights, we propose Adaptive and Robust Channel Hopping (ARCH) protocol, a lightweight receiver-oriented protocol which handles the dynamics of residential environments by reactively channel hopping when channel conditions have degraded. We evaluate our approach through a series of simulations based on real data traces as well as a testbed deployment in real-world apartments. Our results demonstrate that ARCH can reduce the number of packet retransmissions by a median of 42.3% compared to using a single, fixed wireless channel, and can enable up to a 2.2 X improvement in delivery rate on the most unreliable links in our experiment. Due to ARCH\u27s lightweight reactive design, this improvement in reliability is achieved with an average of 6 or fewer channel hops per link per day

Energy-Efficient Low Power Listening for Wireless Sensor Networks in Noisy Environments

Low Power Listening (LPL) is a common MAC-layer technique for reducing energy consumption in wireless sensor networks, where nodes periodically wake up to sample the wireless channel to detect activity. However, LPL is highly susceptible to false wakeups caused by environmental noise being detected as activity on the channel, causing nodes to spuriously wake up in order to receive nonexistent transmissions. In empirical studies in residential environments, we observe that the false wakeup problem can significantly increase a node\u27s duty cycle, compromising the benefit of LPL. We also find that the energy-level threshold used by the Clear Channel Assessment (CCA) mechanism to detect channel activity has a significant impact on the false wakeup rate. We then design AEDP, an adaptive energy detection protocol for LPL, which dynamically adjust a node\u27s CCA threshold to improve network reliability and duty cycle based on application-specified bounds. Empirical experiments in both controlled tests and real-world environments showed AEDP can effectively mitigate the impact of noise on radio duty cycles, while maintaining satisfactory link reliability

ARCH: Practical Channel Hopping for Reliable Home-Area Sensor Networks

Home area networks (HANs) promise to enable sophisticated home automation applications such as smart energy usage and assisted living. However, recent empirical study of HAN reliability in real-world residential environments revealed significant challenges to achieving reliable performance in the face of significant and variable interference from a multitude of coexisting wireless devices. We propose the Adaptive and Robust Channel Hopping (ARCH) protocol: a lightweight receiveroriented protocol which handles the dynamics of residential environments by reactively channel hopping when channel conditions have degraded. ARCH has several key features. First, ARCH is an adaptive protocol that channel-hops based on changes in channel quality observed in real time. Second, ARCH is a distributed protocol that selects channels on a per-link basis, due to the large link-to-link variations in channel quality observed under empirical study. Third, ARCH is designed to be robust and lightweight. ARCH uses a practical hand-shaking approach to handle channel desynchronization and an efficient slidingwindow scheme that does not involve expensive calculations or modeling, and can be reasonably implemented on memoryconstrained wireless sensor platforms. Fourth, ARCH introduces minimal communication overhead for applications where packet acknowledgements are already enabled. We evaluate our approach through real deployment in real-life apartments with residents’ daily activity. Our results demonstrate that ARCH can reduce the number of packet retransmissions by a median of 42.3% compared to using a single, fixed wireless channel, and can enable up to a 2.2 improvement in delivery rate on the most unreliable links in our experiment. Under a multi-hop routing scenario, ARCH achieved an average 31.6% reduction in radio usage, by reducing the ETX along each path by up to 83.6%. Due to ARCH’s lightweight reactive design, most links achieve this improvement in reliability with 10 or fewer channel hops per day

Multi-Channel Reliability and Spectrum Usage in Real Homes: Empirical Studies for Home-Area Sensor Networks

Home area networks (HANs) consisting of wireless sensors have emerged as the enabling technology for important applications such as smart energy and assisted living. A key challenge faced by HANs is maintaining reliable operation in real-world residential environments. This paper presents two in-depth empirical studies on the wireless channels in real homes. The spectrum study analyzes the spectrum usage in the 2.4 GHz band where wireless sensor networks based on the IEEE 802.15.4 standard must coexist with existing wireless devices. We characterize the ambient wireless environment in six apartments through passive spectrum analysis across the entire 2.4 GHz band over seven days in each of the apartments. Notably, we find that the wireless conditions in these residential environments can be much more complex and varied than in a typical office environment. Moreover, while 802.11 signals play a significant role in spectrum usage, there also exist non-negligible noise from non-802.11 devices. The multi-channel link study measures the reliability of different 802.15.4 channels through active probing with motes. We discover that there is not always a persistently reliable channel over 24 hours; that reliability is strongly correlated across adjacent channels; and that link reliability does not exhibit cyclic behavior at daily or weekly timescales. Nevertheless, reliability can be maintained through a small number of channel hops per day, suggesting channel diversity as a key tool for designing robust HANs in residential environments. Our empirical studies provide important guidelines and insights for robust wireless sensor network design in residential environments