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Rice mitochondrial aldehyde dehydrogenase OsALDH2b contributes to tapetal degradation, which is essential for male fertility., Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Although several genes involved in tapetum development have been identified, the molecular basis of tapetum degeneration regulation remains poorly understood. In this study, we identified and characterized the nucleus-encoded, conserved mitochondrial aldehyde dehydrogenase OsALDH2b as a key regulator of tapetum degeneration in rice (Oryza sativa). OsALDH2b was highly expressed in anthers from meiosis to the early microspore stage. Mutation of OsALDH2b resulted in excess malonaldehyde accumulation and earlier programmed cell death in the tapetum, leading to premature tapetum degeneration and abnormal microspore development. These results demonstrate that OsALDH2b negatively regulates tapetal programmed cell death and is required for male reproductive development, providing insights into the regulation of tapetum development in plants.

This study focuses on using limited wind tunnel test records to interpolate surface wind pressures where no measuring taps are arranged or under wind directions that are not measured. Gaussian process regression (GPR), a machine learning method, is first introduced to investigate the issue. For the interpolations of lower-order moments, time histories, higher-order moments, and wind pressure cumulative distribution functions (CDF), several novel GPR-based approaches are proposed. The newly proposed time history interpolation approach can capture the slight variation of wind pressure field with time, owing to the invocation of the time-varying GPR models. Interpolations of skewness, kurtosis, and wind pressure CDFs are attempted for the first time. Taking the surface pressures of a rectangular flat-roof low-rise building as an example, all the proposed approaches are comprehensively discussed, and their interpolation accuracy and superiority are demonstrated by comparing the interpolation results with the measured data and with the outputs from current state-of-the-art approaches.

Genome editing with CRISPR/Cas9 has become an effective tool for gene functionality dissection and crop improvement. Accordingly, we have developed a CRISPR/Cas9 vector system for robust multi-gene editing in plants. Our inventory includes six versatile binary vectors and 12 intermediary sgRNA vectors with different U3 and U6 promoters, to achieve high efficient multi-gene editing, under specific antibiotic selections in both monocot and dicot plants. Additionally, we also have developed an integrated web-based software toolkit, CRISPR-GE, to facilitate the utilization CRISPR/Cas9 in plants. In this protocol, we provide detailed procedures for using the CRISPR/Cas9 system, including target site selection, construction of CRISPR/Cas9 binary vectors, Sanger-sequencing-based genotyping of mutant sites, and troubleshooting for common problems.

Global food security is increasingly challenging in light of population increase, the impact of climate change on crop production, and limited land available for agricultural expansion. Here we outline how genome editing provides excellent and timely methods to optimize crop plants, and argue the urgency for societal acceptance and support.

Use of the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) and Cpf1 systems in plants (Ma et al., 2016; Wang et al., 2017) involves many steps, including the selection of appropriate specific target site(s) that should have no highly homologous sequences as the potential off-target sites in the genome, the design and synthesis of oligonucleotides involving the target sequences, the preparation of expression cassette(s) for the target single guide RNAs (sgRNAs) that provide target sequence specificity, the construction of plant-transformation/expression vector(s), and the transformation of plants, followed by the detection and determination of the targeted mutations in the transgenic plants.

Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA samples. Here we describe protocols for preparation of rice plant gDNAs for various applications including: (1) maxipreps and (2) minipreps of gDNAs for restriction analysis, gene cloning, PCR amplification, genomic sequencing, and genotyping; (3) preparation of megabase-sized nuclear DNA for construction of large-insert genomic libraries and long-range physical mapping; and (4) 96-well-format high-throughput gDNA preparation for PCR-based genotyping. The methods are also suitable for other plant species including dicots. © 2016 by John Wiley & Sons, Inc.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.

A novel algorithm is proposed for simulating univariate non-Gaussian nonstationary processes (NNP) with the specified evolutionary power spectral density (EPSD)/nonstationary auto-correlation function (NACF) and first four-order time-varying marginal moments (TVMMs). The sample realizations of the target NNP are generated as the outputs from a specific time-varying auto-regressive (TVAR) model via filtering the non-Gaussian and nonstationary white noise inputs. These white noise inputs are also non-Gaussian and nonstationary, and their first four-order TVMMs are predetermined using an approach developed herein according to the specified EPSD/NACF and first four-order TVMMs of the outputs. The conventional Johnson transformation is updated to accommodate the nonstationary cases for producing desired white noise inputs. This algorithm is developed from the linear filtering method (LFM), and inherits the simplicity and high efficiency from LFM. It fills the gaps in LFM-based algorithms for simulating NNP. Two numerical examples, i.e., a ground motion acceleration and a downburst velocity, are presented to fully demonstrate the capabilities of the proposed algorithm by comparing the simulation statistics with the targets.

Cloning of coding sequence (CDS) is an important step for gene function research. Here, we reported a simple and efficient strategy for assembling multiple-exon into an intron-free CDS from genomic DNA (gDNA) by an isothermal recombination reaction-based PCR (IRR-PCR) method. As an example, a 2067-bp full-length CDS of the anther-specific expression gene OsABCG15, which is composed of seven exons and six introns, was generated by IRR-PCR using genomic DNA of rice leaf as the template. Actually, this approach can be wildly applied to any DNA sequences assembly to achieve CDS cloning, gene fusion and multiple site-directed mutagenesis in functional genomics studies in vitro.

Bt cotton remains one of the most widely grown biotech crops among smallholder farmers in lower income countries, and numerous studies attest to its advantages. However, the effectiveness of Bt toxin, which depends on many technical constraints, is heterogeneous. In Pakistan, the diffusion of Bt cotton occurred despite a weak regulatory system and without seed quality control; whether or not many varieties sold as Bt are in fact Bt is also questionable. We utilise nationally representative sample data to test the effects of Bt cotton use on productivity. Unlike previous studies, we invoke several indicators of Bt identity: variety name, official approval status, farmer belief, laboratory tests of Bt presence in plant tissue, and biophysical assays measuring Bt effectiveness. Only farmer belief affects cotton productivity in the standard production model, which does not treat Bt appropriately as damage-abating. In the damage control framework, all Bt indicators reduce damage from pests. Biophysical indicators have the largest effect and official approval has the weakest. Findings have implications for impact measurement. For policy-makers, they suggest the need, on ethical and productivity grounds, to improve variety information and monitor variety integrity closer to point of sale.

Few studies have attempted to characterize the pheasant (Phasianus colchicus) immunoglobulin Y (IgY) heavy chain constant region. In the present study, fragments of the pheasant IgY heavy chain constant region were cloned, analyzed, and expressed. The cross-reactivity of IgY or immunoglobulin G (IgG)s with antigens from other vertebrate species was determined using dot-enzyme-linked immunosorbent assay and western blot analysis. Five peptides of the pheasant IgY heavy chain constant region were synthesized to determine its immunoregulatory activity in vitro. The IgY heavy chain constant region from pheasant showed the highest homology with that from chicken (71.2 %) and duck (49.1 %). Phylogenetic analysis for IgY showed that pheasant was closely related to chicken and duck than to any other analyzed vertebrate species. The rabbit anti-chicken IgG showed immunologic cross-reactivity with recombinant proteins of the pheasant IgY heavy chain constant region. Four peptides were able to induce significant up-regulation of interleukin (IL)-1β, IL-4, and interferon-γ in chicken peripheral blood lymphocytes, suggesting a new role of avian IgY in immune regulation.

The role of intellectual property rights (IPRs) has been extensively debated in the literature on technology transfers and agricultural productivity growth in developing countries. However, few studies offer cross-country evidence on how IPRs affect yield growth by incentivising private sector investment in cultivar improvement. We address this knowledge gap by testing technology diffusion patterns for six major crops using a unique dataset for the period 1961–2010 and an Arellano–Bond linear dynamic panel-data estimation approach. Findings indicate that biological and legal forms of IPRs promote yield gap convergence between developed and developing countries, although effects vary by crop.

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

Recently, genome editing technologies have shown great potential in plants. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a new generation of genome editing tool rapidly replacing the earlier zinc finger nucleases and transcription activator-like effector nucleases systems. Indeed, due to its advantages of simplicity and high efficiency, the CRISPR/Cas9-based genome editing system is becoming a powerful tool in plant science research. Here, we introduce the technical features of the plant CRISPR/Cas9-based genome editing system and its applications in plant functional genomics studies and genetic improvement.

Recently, genome editing technologies have shown great potential in plants. The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a new generation of genome editing tool rapidly replacing the earlier zinc finger nucleases and transcription activator-like effector nucleases systems. Indeed, due to its advantages of simplicity and high efficiency, the CRISPR/Cas9-based genome editing system is becoming a powerful tool in plant science research. Here, we introduce the technical features of the plant CRISPR/Cas9-based genome editing system and its applications in plant functional genomics studies and genetic improvement.

Using linear filter method and Johnson transformation, an efficient algorithm for simulating non-Gaussian wind load is developed in this study. Simulations with target first four-order marginal moments and power spectral density are produced by filtering the underlying non-Gaussian white noise that generated by Johnson transformation into non-Gaussian process using linear filter, such as autoregressive (AR) or autoregressive moving average (ARMA) model. For AR and ARMA models, the explicit relationships between the first four-order marginal moments of the input and those of the output are ascertained, thereby the moments of the underlying non-Gaussian white noise input can be calculated. Different from the traditional probability-dependent incompatibility for existing algorithms, a distinctive type of incompatibility which is characterized by spectrum-dependent for the new simulation algorithm is discovered and discussed. In addition, the incompatible ranges of the two types of incompatibilities are compared. We investigated several numerical examples and they demonstrated that the first four-order moments, PSD, and correlation functions of the generations closely matched the targets.

The transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing systems have greatly improved the efficiency for generating targeted mutations in various organisms including plants (Li et al., 2012; Cong et al., 2013; Li et al., 2013; Feng et al., 2014; Ma et al., 2015b; Zhang et al., 2014, 2015). In some plant species, the majority of mutations induced by TALENs and CRISPR/Cas9 systems are in uniform biallelic and heterozygous status in the first transgenic generation, although in some other plant species, chimeric mutations (with three or more allelic edited events within a single individual) may frequently occur (Li et al., 2013; Feng et al., 2014; Zhang et al., 2014, 2015; Ma et al., 2015b).

The recent development of sequence-specific nuclease systems, i.e., TALENs and CRISPR/Cas9, has made genomic targeting easier in many organisms including plants (Li et al., 2012; Cong et al., 2013; Joung and Sander, 2013; Li, et al., 2013; Shan et al., 2013; Liang et al., 2014; Zhang et al., 2014). Mutations induced by CRISPR/Cas9 usually occur around the cleavage sites at three bases upstream of the protospacer-adjacent motif (PAM), producing insertion and deletion of nucleotides. For diploid organisms, such targeted mutations may happen in one or both homologous chromosomes.

A hybrid data and simulation–based (HDSB) approach that incorporates simulated data based on an autoregression (AR) model and an inverse Johnson transformation for estimating wind pressure extremes is introduced. With the target statistical characteristics, i.e., probability density function (PDF) and power spectra density (PSD) in hand, massive simulations of non-Gaussian wind pressure can be carried out, which provides the peak factors or any desired fractile levels for design applications. Existing methods concerning the peak factors of wind pressure are briefly reviewed, and the fundamental basis and the simulation procedure of the HDSB approach are presented. The invocation of the inverse Johnson transformation in the HDSB approach enhances its applicability to the entire Pearson diagram, i.e., for all the softening and hardening stationary non-Gaussian processes with any combination of skewness and kurtosis. The efficacy of the HDSB approach in comparison with existing methods is demonstrate...

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

Summary A vast quantity of measurements of wind-induced non-Gaussian effects on buildings call for the burgeoning development of more advanced extrema estimation approaches for non-Gaussian processes. In this study, a well-directed method for estimating the peak factor and modeling the extrema distribution for non-Gaussian processes is proposed. This method is characterized by using two fitted probability distributions of the parent non-Gaussian process to separately fulfill the estimations of the extrema on long-tail and short-tail sides. In this method, the Johnson transformation is adopted to be the probabilistic model for fitting the parent distribution of the non-Gaussian process due to its superior fitting goodness and universality. For each dataset, two Johnson transformations will be established by two parameter estimation methods to individually estimate the extrema on two sides. Then a Gumbel assumption is applied for conveniently determining the non-Gaussian peak factor. This method is validated through long-duration wind pressure records measured on the model surfaces of a high-rise building in wind tunnel test. The results show that the proposed method is more accurate and robust than many existing ones in estimating peak factors for non-Gaussian wind pressures.