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).