BackgroundSequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and provide an opportunity for comprehensive annotation of TEs. Numerous methods exist for annotation of each class of TEs, but their relative performances have not been systematically compared. Moreover, a comprehensive pipeline is needed to produce a non-redundant library of TEs for species lacking this resource to generate whole-genome TE annotations.ResultsWe benchmark existing programs based on a carefully curated library of rice TEs. We evaluate the performance of methods annotating long terminal repeat (LTR) retrotransposons, terminal inverted repeat (TIR) transposons, short TIR transposons known as miniature inverted transposable elements (MITEs), and Helitrons. Performance metrics include sensitivity, specificity, accuracy, precision, FDR, and F1. Using the most robust programs, we create a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a filtered non-redundant TE library for annotation of structurally intact and fragmented elements. EDTA also deconvolutes nested TE insertions frequently found in highly repetitive genomic regions. Using other model species with curated TE libraries (maize and Drosophila), EDTA is shown to be robust across both plant and animal species.ConclusionsThe benchmarking results and pipeline developed here will greatly facilitate TE annotation in eukaryotic genomes. These annotations will promote a much more in-depth understanding of the diversity and evolution of TEs at both intra- and inter-species levels. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.

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20Sequencing technology and assembly algorithms have matured to the point that high-21 quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse 22 transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for 23 each class of elements with unknown relative performance metrics. We benchmarked existing 24 programs based on a curated library of rice TEs. Using the most robust programs, we created a 25 comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a 26 condensed TE library for annotations of structurally intact and fragmented elements. EDTA is 27 open-source and freely available: https://github.com/oushujun/EDTA. 28 Keywords 29 Transposable element; Annotation; Genome; Benchmarking; Pipeline 30 31Long-read sequencing (e.g., PacBio and Oxford Nanopore) and assembly scaffolding 50 (e.g., Hi-C and BioNano) techniques have progressed rapidly within the last few years. These 51 innovations have been critical for high-quality assembly of the repetitive fraction of genomes. In 52 fact, Ou et al. [8] demonstrated that the assembly contiguity of repetitive sequences in recent 53 long-read assemblies is even better than traditional BAC-based reference genomes. With these 54 developments, inexpensive and high-quality assembly of an entire genome is now possible. 55Knowing where features (i.e., genes, TEs, etc.) exist in a genome assembly is important 56 4 information for using these assemblies for biological findings. However, unlike the relatively 57 straightforward and comprehensive pipelines established for gene annotation [9][10][11], current 58 methods for TE annotation can be piecemeal, inaccurate, and are highly specific to classes of 59 transposable elements. 60Transposable elements fall into two major classes. Class I elements, also known as 61 retrotransposons, use an RNA intermediate in their "copy and paste" mechanism of 62 transposition [12]. Class I elements can be further divided into long terminal repeat (LTR) 63 retrotransposons, as well as those that lack LTRs (non-LTRs), which include long interspersed 64 nuclear elements (LINEs), and short interspersed nuclear elements (SINEs). Structural features 65 of these elements can facilitate automated de novo annotation in a genome assembly. For 66 example, LTR elements have a 5-bp target site duplication (TSD), while non-LTRs have either 67 variable length TSDs or lack TSDs entirely, being instead associated with deletion of flanking 68 sequences upon insertion [13]. There are also standard terminal sequences associated with 69 LTR elements (i.e., 5'-TG…C/G/TA-3' for LTR-Copia and 5'-TG…CA-3' for LTR-Gypsy 70 elements), and non-LTRs often have a terminal poly-A tail at the 3' end of the element (see [14] 71 for a complete description of structural features of each superfamily). 72The second major class of TEs, Class II elements, also known as DNA transposons, use 73 a DNA intermediate in their "cut and paste" mechanism of transposition [15]. As with Class I 74...

We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA doublestrand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFNinduced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.nonhomologous end-joining | gene knockout | alcohol dehydrogenase | chalcone synthase

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.