statgenMPP: an R package implementing an IBD-based mixed model approach for QTL mapping in a wide range of multi-parent populations.

MOTIVATION: Multi-parent populations (MPPs) are popular for QTL mapping because they combine wide genetic diversity in parents with easy control of population structure, but a limited number of software tools for QTL mapping are specifically developed for general MPP designs. RESULTS: We developed an R package called statgenMPP, adopting a unified identity-by-descent (IBD)-based mixed model approach for QTL analysis in MPPs. The package offers easy-to-use functionalities of IBD calculations, mixed model solutions and visualizations for QTL mapping in a wide range of MPP designs, including diallele, nested-association mapping populations, multi-parent advanced genetic inter-cross populations and other complicated MPPs with known crossing schemes. AVAILABILITY AND IMPLEMENTATION: The R package statgenMPP is open-source and freely available on CRAN at https://CRAN.R-project.org/package=statgenMPP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An IBD-based mixed model approach for QTL mapping in multiparental populations.

KEY MESSAGE: The identity-by-descent (IBD)-based mixed model approach introduced in this study can detect quantitative trait loci (QTLs) referring to the parental origin and simultaneously account for multilevel relatedness of individuals within and across families. This unified approach is proved to be a powerful approach for all kinds of multiparental population (MPP) designs. Multiparental populations (MPPs) have become popular for quantitative trait loci (QTL) detection. Tools for QTL mapping in MPPs are mostly developed for specific MPPs and do not generalize well to other MPPs. We present an IBD-based mixed model approach for QTL mapping in all kinds of MPP designs, e.g., diallel, Nested Association Mapping (NAM), and Multiparental Advanced Generation Intercross (MAGIC) designs. The first step is to compute identity-by-descent (IBD) probabilities using a general Hidden Markov model framework, called reconstructing ancestry blocks bit by bit (RABBIT). Next, functions of IBD information are used as design matrices, or genetic predictors, in a mixed model approach to estimate variance components for multiallelic genetic effects associated with parents. Family-specific residual genetic effects are added, and a polygenic effect is structured by kinship relations between individuals. Case studies of simulated diallel, NAM, and MAGIC designs proved that the advanced IBD-based multi-QTL mixed model approach incorporating both kinship relations and family-specific residual variances (IBD.MQMkin_F) is robust across a variety of MPP designs and allele segregation patterns in comparison to a widely used benchmark association mapping method, and in most cases, outperformed or behaved at least as well as other tools developed for specific MPP designs in terms of mapping power and resolution. Successful analyses of real data cases confirmed the wide applicability of our IBD-based mixed model methodology.

Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains.

BACKGROUND: Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers. RESULTS: The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping. We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population. CONCLUSION: The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under "GNU GPL v3". The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl .

Identifying genotype-by-environment interactions in the metabolism of germinating arabidopsis seeds using generalized genetical genomics.

A complex phenotype such as seed germination is the result of several genetic and environmental cues and requires the concerted action of many genes. The use of well-structured recombinant inbred lines in combination with "omics" analysis can help to disentangle the genetic basis of such quantitative traits. This so-called genetical genomics approach can effectively capture both genetic and epistatic interactions. However, to understand how the environment interacts with genomic-encoded information, a better understanding of the perception and processing of environmental signals is needed. In a classical genetical genomics setup, this requires replication of the whole experiment in different environmental conditions. A novel generalized setup overcomes this limitation and includes environmental perturbation within a single experimental design. We developed a dedicated quantitative trait loci mapping procedure to implement this approach and used existing phenotypical data to demonstrate its power. In addition, we studied the genetic regulation of primary metabolism in dry and imbibed Arabidopsis (Arabidopsis thaliana) seeds. In the metabolome, many changes were observed that were under both environmental and genetic controls and their interaction. This concept offers unique reduction of experimental load with minimal compromise of statistical power and is of great potential in the field of systems genetics, which requires a broad understanding of both plasticity and dynamic regulation.

Visualizing the genetic landscape of Arabidopsis seed performance.

Perfect timing of germination is required to encounter optimal conditions for plant survival and is the result of a complex interaction between molecular processes, seed characteristics, and environmental cues. To detangle these processes, we made use of natural genetic variation present in an Arabidopsis (Arabidopsis thaliana) Bayreuth × Shahdara recombinant inbred line population. For a detailed analysis of the germination response, we characterized rate, uniformity, and maximum germination and discuss the added value of such precise measurements. The effects of after-ripening, stratification, and controlled deterioration as well as the effects of salt, mannitol, heat, cold, and abscisic acid (ABA) with and without cold stratification were analyzed for these germination characteristics. Seed morphology (size and length) of both dry and imbibed seeds was quantified by using image analysis. For the overwhelming amount of data produced in this study, we developed new approaches to perform and visualize high-throughput quantitative trait locus (QTL) analysis. We show correlation of trait data, (shared) QTL positions, and epistatic interactions. The detection of similar loci for different stresses indicates that, often, the molecular processes regulating environmental responses converge into similar pathways. Seven major QTL hotspots were confirmed using a heterogeneous inbred family approach. QTLs colocating with previously reported QTLs and well-characterized mutants are discussed. A new connection between dormancy, ABA, and a cripple mucilage formation due to a naturally occurring mutation in the MUCILAGE-MODIFIED2 gene is proposed, and this is an interesting lead for further research on the regulatory role of ABA in mucilage production and its multiple effects on germination parameters.

GERMINATOR: a software package for high-throughput scoring and curve fitting of Arabidopsis seed germination.

Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task that is often prohibitive to the execution of large experiments. In this paper we present the germinator package: a simple, highly cost-efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The germinator package contains three modules: (i) design of experimental setup with various options to replicate and randomize samples; (ii) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (iii) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve-fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of recombinant inbred lines and were able to identify several quantitative trait loci for salt tolerance. Germinator is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.

Network Analysis Prioritizes DEWAX and ICE1 as the Candidate Genes for Major eQTL Hotspots in Seed Germination of Arabidopsis thaliana.

Seed germination is characterized by a constant change of gene expression across different time points. These changes are related to specific processes, which eventually determine the onset of seed germination. To get a better understanding on the regulation of gene expression during seed germination, we performed a quantitative trait locus mapping of gene expression (eQTL) at four important seed germination stages (primary dormant, after-ripened, six-hour after imbibition, and radicle protrusion stage) using Arabidopsis thaliana Bay x Sha recombinant inbred lines (RILs). The mapping displayed the distinctness of the eQTL landscape for each stage. We found several eQTL hotspots across stages associated with the regulation of expression of a large number of genes. Interestingly, an eQTL hotspot on chromosome five collocates with hotspots for phenotypic and metabolic QTL in the same population. Finally, we constructed a gene co-expression network to prioritize the regulatory genes for two major eQTL hotspots. The network analysis prioritizes transcription factors DEWAX and ICE1 as the most likely regulatory genes for the hotspot. Together, we have revealed that the genetic regulation of gene expression is dynamic along the course of seed germination.

Metabolomic analysis of tomato seed germination.

INTRODUCTION: Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development. OBJECTIVE: This study aimed to assess an integrative approach to explore genetics modulating seed metabolism in different developmental stages and the link between seed metabolic- and germination traits. METHODS: We have utilized gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) metabolite profiling to characterize tomato seeds during dry and imbibed stages. We describe, for the first time in tomato, the use of a so-called generalized genetical genomics (GGG) model to study the interaction between genetics, environment and seed metabolism using 100 tomato recombinant inbred lines (RILs) derived from a cross between Solanum lycopersicum and Solanum pimpinellifolium. RESULTS: QTLs were found for over two-thirds of the metabolites within several QTL hotspots. The transition from dry to 6 h imbibed seeds was associated with programmed metabolic switches. Significant correlations varied among individual metabolites and the obtained clusters were significantly enriched for metabolites involved in specific biochemical pathways. CONCLUSIONS: Extensive genetic variation in metabolite abundance was uncovered. Numerous identified genetic regions that coordinate groups of metabolites were detected and these will contain plausible candidate genes. The combined analysis of germination phenotypes and metabolite profiles provides a strong indication for the hypothesis that metabolic composition is related to germination phenotypes and thus to seed performance.

Correlating gene expression to physiological parameters and environmental conditions during cold acclimation of Pinus sylvestris, identification of molecular markers using cDNA microarrays.

Scots pine (Pinus sylvestris L.) seedlings were grown under different conditions (three field locations, two seasons and two climate room regimes), and then analyzed for freezing tolerance of shoots and roots and for transcript abundance in apical buds based on a cDNA microarray containing about 1500 expressed sequence tags (ESTs) from buds of cold-treated Scots pine seedlings. In a climate room providing long daily photoperiods and high temperatures, seedlings did not develop freezing tolerance, whereas seedlings in a climate room set to provide declining temperatures and day lengths developed moderate freezing tolerance. Control seedlings grown outside under field conditions developed full freezing tolerance. Differences in physiological behavior of the different seedling groups, combined with molecular analysis, allowed identification of a large group of genes, expression of which changed during the development of freezing tolerance. Transcript abundance of several of these genes was highly correlated with freezing tolerance in seedlings differing in provenance, field location or age, making them excellent candidate marker genes for molecular tests for freezing tolerance.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation.

Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways.

Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a largely uncharacterized set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover. A number of the target genes have been shown to be expressed in meristems or to be involved in cell wall modifications associated with dividing/growing cells. One of the BBM target genes encodes an ADF/cofilin protein, ACTIN DEPOLYMERIZING FACTOR9 (ADF9). The consequences of BBM:GR activation on the actin cytoskeleton were followed using the GFP:FIMBRIN ACTIN BINDING DOMAIN2 (GFP:FABD) actin marker. Dexamethasone-mediated BBM:GR activation induced dramatic changes in actin organization resulting in the formation of dense actin networks with high turnover rates, a phenotype that is consistent with cells that are rapidly undergoing cytoplasmic reorganization. Together the data suggest that the BBM transcription factor activates a complex network of developmental pathways associated with cell proliferation and growth.

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.).

Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.

Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development.

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.

Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype.

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T-DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.