Fast-forward genetics enabled by new sequencing technologies

New sequencing technologies are dramatically accelerating progress in forward genetics, and the use of such methods for the rapid identification of mutant alleles will be soon routine in many laboratories. A straightforward extension will be the cloning of major-effect genetic variants in crop species. In the near future, it can be expected that mapping by sequencing will become a centerpiece in efforts to discover the genes responsible for quantitative trait loci. The largest impact, however, might come from the use of these strategies to extract genes from non-model, non-crop plants that exhibit heritable variation in important traits. Deployment of such genes to improve crops or engineer microbes that produce valuable compounds heralds a potential paradigm shift for plant biology.     

Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti

An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti . Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA–CheY2 and CheA–CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal β4-α4-β5-α5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting β5 and α5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.     

Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1

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Molecular,genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17‐Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F‐box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col‐FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South‐Eastern Europe approximately 5000 years ago and the FRI‐Col allele to North‐West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The ‘American haplotype’ was identified from several European localities, potentially due to return migration.     

fdrtool: a versatile R package for estimating local and tail area-based false discovery rates

False discovery rate (FDR) methodologies are essential in the study of high-dimensional genomic and proteomic data. The R package 'fdrtool' facilitates such analyses by offering a comprehensive set of procedures for FDR estimation. Its distinctive features include: (i) many different types of test statistics are allowed as input data, such as P-values, z-scores, correlations and t-scores; (ii) simultaneously, both local FDR and tail area-based FDR values are estimated for all test statistics and (iii) empirical null models are fit where possible, thereby taking account of potential over- or underdispersion of the theoretical null. In addition, 'fdrtool' provides readily interpretable graphical output, and can be applied to very large scale (in the order of millions of hypotheses) multiple testing problems. Consequently, 'fdrtool' implements a flexible FDR estimation scheme that is unified across different test statistics and variants of FDR. AVAILABILITY: The program is freely available from the Comprehensive R Archive Network (http://cran.r-project.org/) under the terms of the GNU General Public License (version 3 or later). CONTACT: strimmer@uni-leipzig.de.     

Rapid Identification of a Natural Knockout Allele of ARMADILLO REPEAT-CONTAINING KINESIN1 That Causes Root Hair Branching by Mapping-By-Sequencing

In Arabidopsis (Arabidopsis thaliana), branched root hairs are an indicator of defects in root hair tip growth. Among 62 accessions, one accession (Heiligkreuztal2 [HKT2.4]) displayed branched root hairs, suggesting that this accession carries a mutation in a gene of importance for tip growth. We determined 200- to 300-kb mapping intervals using a mapping-by-sequencing approach of F2 pools from crossings of HKT2.4 with three different accessions. The intersection of these mapping intervals was 80 kb in size featuring not more than 36 HKT2.4-specific single nucleotide polymorphisms, only two of which changed the coding potential of genes. Among them, we identified the causative single nucleotide polymorphism changing a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1. The applied strategies have the potential to complement statistical methods in high-throughput phenotyping studies using different natural accessions to identify causative genes for distinct phenotypes represented by only one or a few accessions.Root hairs are tubular tip outgrowths of single root epidermal cells (trichoblasts). They are an excellent genetic system and serve as a model to study the molecular components regulating tip growth (Carol and Dolan, 2002; Samaj et al., 2004; Lee and Yang, 2008). One of the main regulators of tip growth in root hairs is the small G protein RHO OF PLANTS2 (ROP2; Jones et al., 2002; Payne and Grierson, 2009). ROP2 determines the position of root hairs in incipient epidermal root hair cells and remains localized in the emerging tip during root hair tip growth (Molendijk et al., 2001; Jones et al., 2002). In addition, other factors have been identified to be important for growth and its directionality including phosphoinositides, cytoplasmic [Ca²⁺] gradients and their oscillation, reactive oxygen species, the RAB GTPase homolog A4B, and the cytoskeleton (Foreman et al., 2003; Preuss et al., 2004, 2006; Carol et al., 2005; Thole et al., 2008; Heilmann, 2009).Defects in essential processes for the establishment and maintenance of tip growth lead to deviations in root hair morphology such as branching and waviness (Samaj et al., 2004; Lee and Yang, 2008). Both the microtubules (MTs) and actin are important regulators of tip growth with MTs maintaining one growth point (Bibikova et al., 1999; Miller et al., 1999; Baluska et al., 2000). The latter is evident from the finding that artificially induced [Ca²⁺] gradients can induce additional growth tips when MTs are destroyed by drug treatments (Bibikova et al., 1999).Although the genetic and molecular analysis revealed a well-understood working model for root hair growth, little is known about the natural variation of the underlying processes. Which are the adaptive processes of relevance to specific environmental cues and which have already been selected for in natural accessions? One way to address this question is to link genotype and phenotype by association mapping using various Arabidopsis (Arabidopsis thaliana) accessions. This is greatly facilitated by the 1001 Genomes Project (http://1001genomes.org), providing an increasing number of sequenced accessions. In some cases, phenotypes are only found in one or a few accessions. When the minor allele frequency (AF) is low, the identification of such rare causative alleles with genome-wide association mapping studies is challenging because they cannot be discriminated from false positives (e.g. sequencing errors or synthetic associations; Korte and Farlow, 2013), they are not detectable because of chosen thresholds, or they do not support a statistically significant value (Cantor et al., 2010). Other time-consuming approaches with low mapping resolution, such as quantitative trait loci mapping, need to be followed to identify the causative gene. For this, mapping-by-sequencing, which was originally developed for the identification of mutagen-induced changes in model species (Schneeberger et al., 2009b), can help to rapidly identify causal polymorphisms including nonmodel and nonreference strains (Nordström et al., 2013; Takagi et al., 2013). The resolution of mapping-by-sequencing experiments in Arabidopsis mapping populations is typically between multiple hundreds of kilobase pairs up to a few megabase pairs (James et al., 2013). Although intervals of this size allow the identification of causal mutations in forward genetic screens, they are problematic for the analysis of diverse Arabidopsis accessions because the single nucleotide polymorphism (SNP) density is very high; consequently, hundreds of polymorphisms have to be considered.Here we report on a modification of the mapping-by-sequencing strategy providing a shortcut from distinct, monogenic accession-specific phenotypes to the causative SNP. When studying root hair morphology in 62 accessions for which the genome sequences were released by the 1001 Genomes Project (Cao et al., 2011), we found one accession (Heiligkreuztal2 [HKT2.4]) in which almost all root hairs were branched. To identify the causative gene, we used an approach based on mapping-by-sequencing. Instead of one outcross, we used outcrosses with three different accessions. We selected F2 seedlings exhibiting the distinct, monogenic, recessive root hair branching phenotype for sequencing. Combining the intersection of the three resulting mapping intervals with a selection for accession-specific SNPs revealed two primary candidate genes responsible for the root phenotype. We demonstrate that the causative SNP renders a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1 (ARK1) inactive and therefore leads to a defective ARK1/MORPHOGENESIS OF ROOT HAIR2 (MRH2) protein that is thought to coordinate actin microfilaments and MTs during tip growth of root hairs (Yang et al., 2007; Yoo and Blancaflor, 2013).     

Genome-wide comparison of nucleotide-binding site-leucine-rich repeat-encoding genes in Arabidopsis

Plants, like animals, use several lines of defense against pathogen attack. Prominent among genes that confer disease resistance are those encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) proteins. Likely due to selection pressures caused by pathogens, NB-LRR genes are the most variable gene family in plants, but there appear to be species-specific limits to the number of NB-LRR genes in a genome. Allelic diversity within an individual is also increased by obligatory outcrossing, which leads to genome-wide heterozygosity. In this study, we compared the NB-LRR gene complement of the selfer Arabidopsis thaliana and its outcrossing close relative Arabidopsis lyrata. We then complemented and contrasted the interspecific patterns with studies of NB-LRR diversity within A. thaliana. Three important insights are as follows: (1) that both species have similar numbers of NB-LRR genes; (2) that loci with single NB-LRR genes are less variable than tandem arrays; and (3) that presence-absence polymorphisms within A. thaliana are not strongly correlated with the presence or absence of orthologs in A. lyrata. Although A. thaliana individuals are mostly homozygous and thus potentially less likely to suffer from aberrant interaction of NB-LRR proteins with newly introduced alleles, the number of NB-LRR genes is similar to that in A. lyrata. In intraspecific and interspecific comparisons, NB-LRR genes are also more variable than receptor-like protein genes. Finally, in contrast to Drosophila, there is a clearly positive relationship between interspecific divergence and intraspecific polymorphisms.