The genetic and molecular basis of haploinsufficiency in flowering plants.

In diploid organisms, haploinsufficiency can be defined as the requirement for more than one fully functional copy of a gene. In contrast to most genes, whose loss-of-function alleles are recessive, loss-of-function alleles of haploinsufficient genes are dominant. However, forward and reverse genetic screens are biased toward obtaining recessive, loss-of-function mutations, and therefore, dominant mutations of all types are underrepresented in mutant collections. Despite this underrepresentation, haploinsufficient loci have intriguing implications for studies of genome evolution, gene dosage, stability of protein complexes, genetic redundancy, and gene expression. Here we review examples of haploinsufficiency in flowering plants and describe the underlying molecular mechanisms and evolutionary forces driving haploinsufficiency. Finally, we discuss the masking of haploinsufficiency by genetic redundancy, a widespread phenomenon among angiosperms.

Versatile mapping-by-sequencing with Easymap v.2.

Mapping-by-sequencing combines Next Generation Sequencing (NGS) with classical genetic mapping by linkage analysis to establish gene-to-phenotype relationships. Although numerous tools have been developed to analyze NGS datasets, only a few are available for mapping-by-sequencing. One such tool is Easymap, a versatile, easy-to-use package that performs automated mapping of point mutations and large DNA insertions. Here, we describe Easymap v.2, which also maps small insertion/deletions (InDels), and includes workflows to perform QTL-seq and variant density mapping analyses. Each mapping workflow can accommodate different experimental designs, including outcrossing and backcrossing, F2, M2, and M3 mapping populations, chemically induced mutation and natural variant mapping, input files containing single-end or paired-end reads of genomic or complementary DNA sequences, and alternative control sample files in FASTQ and VCF formats. Easymap v.2 can also be used as a variant analyzer in the absence of a mapping algorithm and includes a multi-threading option.

Mapping-by-Sequencing of Point and Insertional Mutations with Easymap.

Random mutagenesis followed by screening for phenotypes of interest is a widely used strategy for genetic dissection of biological pathways; however, identifying the causal gene traditionally required time-consuming mapping approaches based on iterative linkage analysis. Mapping-by-sequencing accelerates this process, efficiently linking the phenotype of a mutant to a narrow candidate genomic region, using next-generation sequencing (NGS) data from a mapping population segregating for the mutant phenotype. To enable researchers at any bioinformatics skill level to conduct mapping-by-sequencing, we developed the Easymap mapping software. In this protocol we break down the steps involved in mapping-by-sequencing. First, we describe different ways of obtaining a mapping population and the steps used to generate NGS data. Next, we show how to analyze the NGS data using Easymap and how to obtain a list of candidate mutations, along with comprehensive information for assessing the potential causality of each candidate. Thus, this protocol enables the user to conduct mapping-by-sequencing using Easymap, facilitating the identification of causal loci for a mutant phenotype of interest.

Easymap: A User-Friendly Software Package for Rapid Mapping-by-Sequencing of Point Mutations and Large Insertions.

Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existing software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethyl methanesulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results. Easymap is available at http://genetics.edu.umh.es/resources/easymap; its Quickstart Installation Guide details the recommended procedure for installation.

Next-generation forward genetic screens: using simulated data to improve the design of mapping-by-sequencing experiments in Arabidopsis.

Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.

Wound signaling of regenerative cell reprogramming.

Plants are sessile organisms that must deal with various threats resulting in tissue damage, such as herbivore feeding, and physical wounding by wind, snow or crushing by animals. During wound healing, phytohormone crosstalk orchestrates cellular regeneration through the establishment of tissue-specific asymmetries. In turn, hormone-regulated transcription factors and their downstream targets coordinate cellular responses, including dedifferentiation, cell cycle reactivation and vascular regeneration. By comparing different examples of wound-induced tissue regeneration in the model plant Arabidopsis thaliana, a number of key regulators of developmental plasticity of plant cells have been identified. We present the relevance of these findings and of the dynamic establishment of differential auxin gradients for cell reprogramming after wounding.