植物科学

Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.

Relative chromosome dosage, i.e., increases or decreases in the number of copies of specific chromosome regions in one sample versus another, can be determined using aligned read-counts from Illumina sequencing (Henry et al., 2010). The following protocol was used to identify the different classes of aneuploids that result from uniparental genome elimination in Arabidopsis thaliana, including chromosomes that have undergone chromothripsis (Tan et al., 2015). Uniparental genome elimination results in the production of haploid progeny from crosses to specific strains called “haploid inducers” (Ravi et al., 2014). On the other hand, chromothripsis, which was first discovered in cancer genomes, is a phenomenon that results in clustered, highly rearranged chromosomes. In plants, chromothripsis has been observed as a result of genome elimination (Tan et al., 2015). Detecting variation in chromosome dosage has multiple applications beside those linked to genome elimination. For example, a dosage variant population of poplar hybrids was created by gamma-irradiation of pollen grains. Hundreds of dosage lesions, insertions and deletions, were identified using this technique and provide a way to associate loci with the phenotypic consequences observed in this population (Henry et al., 2015).

This method has been successfully used to detect changes in chromosome dosage in many different species, including Arabidopsis thaliana (Tan et al., 2015), Arabidopsis suecica (Ravi et al., 2014), rice (Henry et al., 2010) and poplar (Henry et al., 2015). It is important to note that dosage plots always indicate dosage variation relative to the control sample used (Note 1). Therefore, this approach is not suitable to detect ploidy variants (diploid vs triploid, for example). Similarly, this technique does not allow the detection of balanced chromosomal rearrangements such as reciprocal translocations.

The construction of a physical collection of open reading frames (ORFeomes) for genes of any model organism is a useful tool for the exploration of gene function, gene regulation, and protein-protein interaction. Here we describe in detail a protocol that has been used to develop the first collection of transcription factor (TF) and co-regulator (CR) open reading frames (TFome) in maize (Burdo et al., 2014). This TFome is being used to establish the architecture of gene regulatory networks (GRNs) responsible for the control of transcription of all genes in an organism. The protocol outlined here describes how to proceed when only an incomplete genome with partial annotation is available. TFome clones are made in a recombination-ready vector of the Gateway^? system, allowing for the facile transfer of the ORFs to other Gateway^?-compatible vectors, such as those suitable for expression in other host species. Although this protocol was developed for the maize TFome it can readily be applied to the generation of complete ORFeome collections in other eukaryotic species.

[Protocol overview] An important aspect of successful TFome generation is the initial effort spent to establish a reliable set of gene models so that they can be subsequently amplified or synthesized. An actual TFome construction protocol for a particular species will depend on available resources such as a full-length cDNA (flcDNA) collection and a reliable reference genome (Figure 1).

In the case of maize, a flcDNA collection and a draft genome was available, but the former provided only 30% of the needed clones, and the latter contained gaps and some erroneous gene models. In order to develop a near-complete set of target gene models for maize TFs, a bioinformatics pipeline was developed as described by Yilmaz et al. (2009). In brief, a two-pronged search process was developed. The first involved making a collection of protein sequences of TFs in other species and available from databases such as PlantTFDB, PlnTFDB and DBDTF. These sequences were then used to search gene models from the draft maize genome using BLASTP. The second process involved developing a collection of domains that define TF families and that are mostly annotated in the PFAM database (Finn et al., 2014). These domains were then used to search the draft maize genome using BLASTX. The number of TF families that exist and their naming is subject to change as new members are discovered and studied. Table 1 provides a list of known TF families with alternative names along with the respective PFAM domains whose presence or absence defines each TF family. HMM models for each domain can be obtained from the PFAM database (pfam.xfam.org). Following the BLAST search, redundant models are eliminated and then based on the TF motifs present in each gene model, gene models are assigned to a TF or Co-Regulator (CR) family according to the criteria specified in Table 1. Lastly, it is recommended to set up a database to store information on each TF family. The GRASSIUS (www.grassius.org) website was established to access the stored information on TF gene models for maize, sorghum, rice, Brachypodium, sugarcane and other grasses (Burdo et al., 2014). In the following section, an assumption is made that at least a draft genome or draft transcriptome is available and that a set of gene models is available that have been determined ab initio or with additional manual annotation. Familiarity with the use of PERL scripts is advantageous for the gene model assembly phase.


Figure 1. Flowchart for the generation of a TFome project. Flowchart outlining the general strategy for template identification, PCR amplification and cloning of transcription factor (TF) full length (FL) open reading frames (ORFs). (modified from Burdo et al., 2014)

This protocol describes whole genome bisulfite-sequencing library preparation from plant tissue and subsequent data analysis. Allele-specific methylation analysis and genome-wide identification of differentially methylated regions are additional features of the analysis procedure.

Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole exome of barley. The Exome Library is provided as a liquid array containing biotinylated probes (Roche/NimbleGen). Subsequently, genomic shotgun DNA fragments hybridized to the Exome Library are affinity-purified using streptavidin coated magnetic beads. The captured library is PCR-amplified and sequenced using high-throughput short read sequencing-by-synthesis.

DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.