Optical and physical mapping with local finishing enables megabase-scale resolution of agronomically important regions in the wheat genome.

BACKGROUND: Numerous scaffold-level sequences for wheat are now being released and, in this context, we report on a strategy for improving the overall assembly to a level comparable to that of the human genome. RESULTS: Using chromosome 7A of wheat as a model, sequence-finished megabase-scale sections of this chromosome were established by combining a new independent assembly using a bacterial artificial chromosome (BAC)-based physical map, BAC pool paired-end sequencing, chromosome-arm-specific mate-pair sequencing and Bionano optical mapping with the International Wheat Genome Sequencing Consortium RefSeq v1.0 sequence and its underlying raw data. The combined assembly results in 18 super-scaffolds across the chromosome. The value of finished genome regions is demonstrated for two approximately 2.5 Mb regions associated with yield and the grain quality phenotype of fructan carbohydrate grain levels. In addition, the 50 Mb centromere region analysis incorporates cytological data highlighting the importance of non-sequence data in the assembly of this complex genome region. CONCLUSIONS: Sufficient genome sequence information is shown to now be available for the wheat community to produce sequence-finished releases of each chromosome of the reference genome. The high-level completion identified that an array of seven fructosyl transferase genes underpins grain quality and that yield attributes are affected by five F-box-only-protein-ubiquitin ligase domain and four root-specific lipid transfer domain genes. The completed sequence also includes the centromere.

Approaches in Characterizing Genetic Structure and Mapping in a Rice Multiparental Population.

Multi-parent Advanced Generation Intercross (MAGIC) populations are fast becoming mainstream tools for research and breeding, along with the technology and tools for analysis. This paper demonstrates the analysis of a rice MAGIC population from data filtering to imputation and processing of genetic data to characterizing genomic structure, and finally quantitative trait loci (QTL) mapping. In this study, 1316 S6:8 indica MAGIC (MI) lines and the eight founders were sequenced using Genotyping by Sequencing (GBS). As the GBS approach often includes missing data, the first step was to impute the missing SNPs. The observable number of recombinations in the population was then explored. Based on this case study, a general outline of procedures for a MAGIC analysis workflow is provided, as well as for QTL mapping of agronomic traits and biotic and abiotic stress, using the results from both association and interval mapping approaches. QTL for agronomic traits (yield, flowering time, and plant height), physical (grain length and grain width) and cooking properties (amylose content) of the rice grain, abiotic stress (submergence tolerance), and biotic stress (brown spot disease) were mapped. Through presenting this extensive analysis in the MI population in rice, we highlight important considerations when choosing analytical approaches. The methods and results reported in this paper will provide a guide to future genetic analysis methods applied to multi-parent populations.

Transcriptomic analysis of wheat near-isogenic lines identifies PM19-A1 and A2 as candidates for a major dormancy QTL.

BACKGROUND: Next-generation sequencing technologies provide new opportunities to identify the genetic components responsible for trait variation. However, in species with large polyploid genomes, such as bread wheat, the ability to rapidly identify genes underlying quantitative trait loci (QTL) remains non-trivial. To overcome this, we introduce a novel pipeline that analyses, by RNA-sequencing, multiple near-isogenic lines segregating for a targeted QTL. RESULTS: We use this approach to characterize a major and widely utilized seed dormancy QTL located on chromosome 4AL. It exploits the power and mapping resolution afforded by large multi-parent mapping populations, whilst reducing complexity by using multi-allelic contrasts at the targeted QTL region. Our approach identifies two adjacent candidate genes within the QTL region belonging to the ABA-induced Wheat Plasma Membrane 19 family. One of them, PM19-A1, is highly expressed during grain maturation in dormant genotypes. The second, PM19-A2, shows changes in sequence causing several amino acid alterations between dormant and non-dormant genotypes. We confirm that PM19 genes are positive regulators of seed dormancy. CONCLUSIONS: The efficient identification of these strong candidates demonstrates the utility of our transcriptomic pipeline for rapid QTL to gene mapping. By using this approach we are able to provide a comprehensive genetic analysis of the major source of grain dormancy in wheat. Further analysis across a diverse panel of bread and durum wheats indicates that this important dormancy QTL predates hexaploid wheat. The use of these genes by wheat breeders could assist in the elimination of pre-harvest sprouting in wheat.

MAGIC populations in crops: current status and future prospects.

KEY MESSAGE: MAGIC populations present novel challenges and opportunities in crops due to their complex pedigree structure. They offer great potential both for dissecting genomic structure and for improving breeding populations. The past decade has seen the rise of multiparental populations as a study design offering great advantages for genetic studies in plants. The genetic diversity of multiple parents, recombined over several generations, generates a genetic resource population with large phenotypic diversity suitable for high-resolution trait mapping. While there are many variations on the general design, this review focuses on populations where the parents have all been inter-mated, typically termed Multi-parent Advanced Generation Intercrosses (MAGIC). Such populations have already been created in model animals and plants, and are emerging in many crop species. However, there has been little consideration of the full range of factors which create novel challenges for design and analysis in these populations. We will present brief descriptions of large MAGIC crop studies currently in progress to motivate discussion of population construction, efficient experimental design, and genetic analysis in these populations. In addition, we will highlight some recent achievements and discuss the opportunities and advantages to exploit the unique structure of these resources post-QTL analysis for gene discovery.

ELF3 controls thermoresponsive growth in Arabidopsis.

Plant development is highly responsive to ambient temperature, and this trait has been linked to the ability of plants to adapt to climate change. The mechanisms by which natural populations modulate their thermoresponsiveness are not known. To address this, we surveyed Arabidopsis accessions for variation in thermal responsiveness of elongation growth and mapped the corresponding loci. We find that the transcriptional regulator EARLY FLOWERING3 (ELF3) controls elongation growth in response to temperature. Through a combination of modeling and experiments, we show that high temperature relieves the gating of growth at night, highlighting the importance of temperature-dependent repressors of growth. ELF3 gating of transcriptional targets responds rapidly and reversibly to changes in temperature. We show that the binding of ELF3 to target promoters is temperature dependent, suggesting a mechanism where temperature directly controls ELF3 activity.

The surprising benefit of passive-aggressive behaviour at Christmas parties: being crowned king of the crackers.

OBJECTIVE: To test the effects of technique and attitude in pulling Christmas crackers. DESIGN, SETTING AND PARTICIPANTS: A binomial trial conducted at a Christmas-in-July dinner party involving five anonymous dinner guests, including two of the authors. MAIN OUTCOME MEASURE: Number of wins achieved by different strategies, with a win defined as securing the larger portion of the cracker. RESULTS: The previously "guaranteed" strategy for victory, employing a downwards angle towards the puller, failed to differentiate itself from random chance (win rate, 6/15; probability of winning, 0.40; 95% CI, 0.15-0.65). A novel passive-aggressive strategy, in which one individual just holds on without pulling, provided a significant advantage (win rate, 11/12; probability of winning, 0.92; 95% CI, 0.76-1.00). CONCLUSION: The passive-aggressive strategy of failing to pull has a high rate of success at winning Christmas crackers; however, excessive adoption of this approach will result in a complete failure, with no winners at all.

QTLs for tolerance of drought and breeding for tolerance of abiotic and biotic stress: an integrated approach.

BACKGROUND: The coupling of biotic and abiotic stresses leads to high yield losses in rainfed rice (Oryza sativa L.) growing areas. While several studies target these stresses independently, breeding strategies to combat multiple stresses seldom exist. This study reports an integrated strategy that combines QTL mapping and phenotypic selection to develop rice lines with high grain yield (GY) under drought stress and non-stress conditions, and tolerance of rice blast. METHODOLOGY: A blast-tolerant BC2F3-derived population was developed from the cross of tropical japonica cultivar Moroberekan (blast- and drought-tolerant) and high-yielding indica variety Swarna (blast- and drought-susceptible) through phenotypic selection for blast tolerance at the BC2F2 generation. The population was studied for segregation distortion patterns and QTLs for GY under drought were identified along with study of epistatic interactions for the trait. RESULTS: Segregation distortion, in favour of Moroberekan, was observed at 50 of the 59 loci. Majority of these marker loci co-localized with known QTLs for blast tolerance or NBS-LRR disease resistance genes. Despite the presence of segregation distortion, high variation for DTF, PH and GY was observed and several QTLs were identified under drought stress and non-stress conditions for the three traits. Epistatic interactions were also detected for GY which explained a large proportion of phenotypic variance observed in the population. CONCLUSIONS: This strategy allowed us to identify QTLs for GY along with rapid development of high-yielding purelines tolerant to blast and drought with considerably reduced efforts. Apart from this, it also allowed us to study the effects of the selection cycle for blast tolerance. The developed lines were screened at IRRI and in the target environment, and drought and blast tolerant lines with high yield were identified. With tolerance to two major stresses and high yield potential, these lines may provide yield stability in rainfed rice areas.

Computationally efficient map construction in the presence of segregation distortion.

KEY MESSAGE: We present a novel estimator for map construction in the presence of segregation distortion which is highly computationally efficient. For multi-parental designs this estimator outperforms methods that do not account for segregation distortion, at no extra computational cost. Inclusion of genetic markers exhibiting segregation distortion in a linkage map can result in biased estimates of genetic distance and distortion of map positions. Removal of distorted markers is hence a typical filtering criterion; however, this may result in exclusion of biologically interesting regions of the genome such as introgressions and translocations. Estimation of additional parameters characterizing the distortion is computationally slow, as it relies on estimation via the Expectation Maximization algorithm or a higher dimensional numerical optimisation. We propose a robust M-estimator (RM) capable of handling tens of thousands of distorted markers from a single linkage group. We show via simulation that for multi-parental designs the RM estimator can perform much better than uncorrected estimation, at no extra computational cost. We then apply the RM estimator to chromosome 2B in wheat in a multi-parent population segregating for the Sr36 introgression, a known transmission distorter. The resulting map contains over 700 markers, and is consistent with maps constructed from crosses which do not exhibit segregation distortion.

Characterizing uncertainty in high-density maps from multiparental populations.

Multiparental populations are of considerable interest in high-density genetic mapping due to their increased levels of polymorphism and recombination relative to biparental populations. However, errors in map construction can have significant impact on QTL discovery in later stages of analysis, and few methods have been developed to quantify the uncertainty attached to the reported order of markers or intermarker distances. Current methods are computationally intensive or limited to assessing uncertainty only for order or distance, but not both simultaneously. We derive the asymptotic joint distribution of maximum composite likelihood estimators for intermarker distances. This approach allows us to construct hypothesis tests and confidence intervals for simultaneously assessing marker-order instability and distance uncertainty. We investigate the effects of marker density, population size, and founder distribution patterns on map confidence in multiparental populations through simulations. Using these data, we provide guidelines on sample sizes necessary to map markers at sub-centimorgan densities with high certainty. We apply these approaches to data from a bread wheat Multiparent Advanced Generation Inter-Cross (MAGIC) population genotyped using the Illumina 9K SNP chip to assess regions of uncertainty and validate them against the recently released pseudomolecule for the wheat chromosome 3B.

Efficient imputation of missing markers in low-coverage genotyping-by-sequencing data from multiparental crosses.

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.

Delay discounting, impulsiveness, and addiction severity in opioid-dependent patients.

Individuals who abuse drugs show higher delay discounting (DD) rate and impulsiveness scores compared with controls; however, it is unclear if DD rate covaries with severity of the addiction or if an individual's discounting rate can be changed by effective substance abuse treatment. This study compared methadone maintenance treatment (MMT) patients (n = 30) who had not used illegal drugs for 2 years with drug-using MMT patients (n = 30) and controls (n = 25) in terms of addiction severity, DD rate, and impulsiveness. Methadone patients abstinent from illegal drugs scored significantly lower on a number of addiction severity measures than the drug-using methadone patients. In addition, both groups of MMT patients showed significantly higher rates of DD and impulsiveness than the control group; however, no differences in DD rate or impulsiveness were found between the groups of patients. Results suggest that DD rate and impulsiveness may not covary with indicators of addiction severity in MMT patients.

R/mpMap: a computational platform for the genetic analysis of multiparent recombinant inbred lines.

UNLABELLED: Multiparent crosses of recombinant inbred lines provide opportunity to map markers and quantitative trait loci (QTL) with much greater resolution than is possible in biparental crosses. Realizing the full potential of these crosses requires computational tools capable of handling the increased statistical complexity of the analyses. R/mpMap provides a flexible and extensible environment, which interfaces easily with other packages to satisfy this demand. Functions in the package encompass simulation, marker map construction, haplotype reconstruction and QTL mapping. We demonstrate the easy-to-use features of mpMap through a simulated data example. AVAILABILITY: www.cmis.csiro.au/mpMap.

Look before you leap: a new approach to mapping QTL.

In this paper, we present an innovative and powerful approach for mapping quantitative trait loci (QTL) in experimental populations. This deviates from the traditional approach of (composite) interval mapping which uses a QTL profile to simultaneously determine the number and location of QTL. Instead, we look before we leap by employing separate detection and localization stages. In the detection stage, we use an iterative variable selection process coupled with permutation to identify the number and synteny of QTL. In the localization stage, we position the detected QTL through a series of one-dimensional interval mapping scans. Results from a detailed simulation study and real analysis of wheat data are presented. We achieve impressive increases in the power of QTL detection compared to composite interval mapping. We also accurately estimate the size and position of QTL. An R library, DLMap, implements the methods described here and is freely available from CRAN ( http://cran.r-project.org/ ).

Interleukin-1 polymorphisms are associated with the inflammatory response in human muscle to acute resistance exercise.

Inflammation appears to play an important role in the repair and regeneration of skeletal muscle after damage. We tested the hypothesis that the severity of the inflammatory response in muscle after an acute bout of resistance exercise is associated with single nucleotide polymorphisms (SNPs) previously shown to alter interleukin-1 (IL-1) activity. Using a double-blind prospective design, sedentary young men were screened (n = 100) for enrolment (n = 24) based upon having 1 of 4 haplotype patterns composed of five polymorphic sites in the IL-1 gene cluster: IL-1A (+4845), IL-1B (+3954), IL-1B (-511), IL-1B (-3737) and IL-1RN (+2018). Subjects performed a standard bout of resistance leg exercise and vastus lateralis biopsies were obtained pre-, and at 24, and 72 h post-exercise. Inflammatory marker mRNAs (IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha)) and the number of CD68(+) macrophages were quantified. Considerable variation was observed in the expression of these gene products between subjects. At 72 h post-exercise, IL-1beta had increased in a number of subjects (n = 10) and decreased (n = 4) or did not change (n = 10) in others. Inflammatory responses were significantly associated with specific haplotype patterns and were also influenced by individual SNPs. Subjects with genotypes 1.1 at IL-1B (+3954) or 2.2 at IL-1B (-3737) had approximately a 2-fold higher median induction of several markers, but no increase in macrophages, suggesting that cytokine gene expression is elevated per macrophage. The IL-1RN (+2018) SNP maximized the response specifically within these groups and was associated with increased macrophage recruitment. This is the first report that IL-1 genotype is associated with the inflammation of skeletal muscle following acute resistance exercise that may potentially affect the adaptations to chronic resistance exercise.