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Population genetics studies of allopolyploid species lag behind those of diploid species because of practical difficulties in analysis of homeologs-duplicated gene copies originating from hybridized parental species. Pool-Seq, i.e. massive parallel sequencing of pooled individuals, has high potential for detecting nucleotide polymorphisms within and among multiple populations; however, its use has been limited to diploid species. We applied Pool-Seq to an allopolyploid species by developing a bioinformatic pipeline that assigns reads to each homeolog as well as to each polymorphic allele within each homeolog. We simultaneously sequenced eight genes from twenty individuals from each of 24 populations, and found over 100 polymorphic sites in each homeolog. For two sites, we estimated allele frequencies using the number of reads and then validated these estimations by making individual-based estimations. Pool-Seq using our bioinformatic pipeline allows efficient evaluation of nucleotide polymorphisms in a large number of individuals, even in allopolyploid species.

Little empirical evidence is available whether allopolyploid species combine or merge adaptations of parental species. The allopolyploid species Arabidopsis kamchatica is a natural hybrid of the diploid parents A. halleri, a heavy metal hyperaccumulator, and A. lyrata, a non-hyperaccumulating species. Zinc and cadmium were measured in native soils and leaf tissues in natural populations, and in hydroponic cultures of A. kamchatica and A. halleri. Pyrosequencing was used to estimate homeolog expression ratios. Soils from human modified sites showed significantly higher Zn concentrations than non-modified sites. Leaf samples of A. kamchatica collected from 40 field localities had > 1,000 µg g-1 Zn in over half of the populations, with significantly higher amounts of Zn concentrations in plants from human modified sites. In addition, serpentine soils were found in two populations. Most genotypes accumulated >3000 µg g-1 of Zn in hydroponic culture with high variability among them. Genes involved in hyperaccumulation showed a bias in the halleri-derived homeolog. A. kamchatica has retained constitutive hyperaccumulation ability inherited from A. halleri. Our field and experimental data provides a compelling example in which the inheritance of genetic toolkits for soil adaptations likely contributed to the habitat expansion of an allopolyploid species.

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

Plant defense inducers that mimic functions of the plant immune hormone salicylic acid (SA) often affect plant growth. Although benzothiadiazole (BTH), a synthetic analog of SA, has been widely used to protect crops from diseases by inducing plant defense responses, we recently demonstrated that SA, but not BTH, confers resistance against Rhizoctonia solani, the causal agent of sheath blight disease, in Brachypodium distachyon. Here, we demonstrated that BTH compromised the resistance of Bd3-1 and Gaz4, the two sheath blight-resistant accessions of B. distachyon, which activate SA-dependent signaling following challenge by R. solani. Moreover, upon analyzing our published RNA-seq data from B. distachyon treated with SA or BTH, we found that BTH specifically induces expression of genes related to chloroplast function and jasmonic acid (JA) signaling, suggesting that BTH attenuates R. solani resistance by perturbing growth-defense trade-offs and/or by inducing a JA response that may increase susceptibility to R. solani. Our findings demonstrated that BTH does not work as a simple mimic of SA in B. distachyon, and consequently may presumably cause unfavorable side effects through the transcriptional alteration, particularly with respect to R. solani resistance.

Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated “multiplex PCR targeted amplicon sequencing (MTA-seq),” and applied it to the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data with that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.

This protocol provides an useful technique for detecting allele frequencies in the cDNA sample

We report the comprehensive identification of periodic genes and their network inference, based on a gene co-expression analysis and an Auto-Regressive eXogenous (ARX) model with a group smoothly clipped absolute deviation (SCAD) method using a time-series transcriptome dataset in a model grass, Brachypodium distachyon. To reveal the diurnal changes in the transcriptome in B. distachyon, we performed RNA-seq analysis of its leaves sampled through a diurnal cycle of over 48 h at 4 h intervals using three biological replications, and identified 3,621 periodic genes through our wavelet analysis. The expression data are feasible to infer network sparsity based on ARX models. We found that genes involved in biological processes such as transcriptional regulation, protein degradation, and post-transcriptional modification and photosynthesis are significantly enriched in the periodic genes, suggesting that these processes might be regulated by circadian rhythm in B. distachyon. On the basis of the time-series expression patterns of the periodic genes, we constructed a chronological gene co-expression network and identified putative transcription factors encoding genes that might be involved in the time-specific regulatory transcriptional network. Moreover, we inferred a transcriptional network composed of the periodic genes in B. distachyon, aiming to identify genes associated with other genes through variable selection by grouping time points for each gene. Based on the ARX model with the group SCAD regularization using our time-series expression datasets of the periodic genes, we constructed gene networks and found that the networks represent typical scale-free structure. Our findings demonstrate that the diurnal changes in the transcriptome in B. distachyon leaves have a sparse network structure, demonstrating the spatiotemporal gene regulatory network over the cyclic phase transitions in B. distachyon diurnal growth.

Seed priming is a commercially used technique for improving seed performance including germination. However, the treatment sometimes reduces seed longevity as a side effect, limiting the storable period or longevity of the seeds. To overcome this problem, molecular mechanisms involved in the loss of seed longevity during priming were analyzed using natural variations of Arabidopsis thaliana. We found that the Est-1 accession retained longevity for longer after priming compared to the reference accession Col-0. QTL analysis using 279 recombinant inbred lines (RILs) derived from the Est-1 × Col-0 detected three QTL regions associated with the loss of seed longevity during priming. Bulked transcriptome analysis (RNA-Seq with bulked RIL populations) revealed that genes related to brassinosteroid (BR) biosynthesis/signaling and cell wall modification were highly expressed in primed seeds with shorter longevity. After priming, BR-deficient mutants cyp85a1/a2 and det2 showed significantly longer longevity than the wild type (WT). Moreover, tetrazolium staining indicated that mutant seed coats were less permeable after priming than those of WT. We suggest that the loss of seed longevity in primed seed is due to increased seed coat permeability, which is positively regulated, at least partly, via BR signaling.

Background Allopolyploid plants often show wider environmental tolerances than their ancestors; this is expected to be due to the merger of multiple distinct genomes with a fixed heterozygosity. The complex homoeologous gene expression could have been evolutionarily advantageous for the adaptation of allopolyploid plants. Despite multiple previous studies reporting homoeolog-specific gene expression in allopolyploid species, there are no clear examples of homoeolog-specific function in acclimation to a long-term stress condition. Results We found that the allopolyploid grass Brachypodium hybridum and its ancestor Brachypodium stacei show long-term heat stress tolerance, unlike its other ancestor, Brachypodium distachyon. To understand the physiological traits of B. hybridum, we compared the transcriptome of the 3 Brachypodium species grown under normal and heat stress conditions. We found that the expression patterns of approximately 26% and approximately 38% of the homoeolog groups in B. hybridum changed toward nonadditive expression and nonancestral expression, respectively, under normal condition. Moreover, we found that B. distachyon showed similar expression patterns between normal and heat stress conditions, whereas B. hybridum and B. stacei significantly altered their transcriptome in response to heat after 3 days of stress exposure, and homoeologs that were inherited from B. stacei may have contributed to the transcriptional stress response to heat in B. hybridum. After 15 days of heat exposure, B. hybridum and B. stacei maintained transcriptional states similar to those under normal conditions. These results suggest that an earlier response to heat that was specific to homoeologs originating from B. stacei contributed to cellular homeostasis under long-term heat stress in B. hybridum. Conclusions Our results provide insights into different regulatory events of the homoeo-transcriptome that are associated with stress acclimation in allopolyploid plants.

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

Background Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. Results We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. Conclusion We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0749-9) contains supplementary material, which is available to authorized users.

Food security has emerged as an urgent concern because of the rising world population. To meet the food demands of the near future, it is required to improve the productivity of various crops, not just of staple food crops. The genetic diversity among plant populations in a given species allows the plants to adapt to various environmental conditions. Such diversity could therefore yield valuable traits that could overcome the food-security challenges. To explore genetic diversity comprehensively and to rapidly identify useful genes and/or allele, advanced high-throughput sequencing techniques, also called next-generation sequencing (NGS) technologies, have been developed. These provide practical solutions to the challenges in crop genomics. Here, we review various sources of genetic diversity in plants, newly developed genetic diversity-mining tools synergized with NGS techniques, and related genetic approaches such as quantitative trait locus analysis and genome-wide association study.

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 degrees C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2+/-24.7 microl h(-1) g(-1)) and post-thermogenic (50.5+/-30.4 microl h(-1) g(-1)) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.

The natural occurrence of temperature-triggered and light-independent thermogenic oscillation in the spadix of skunk cabbage, Symplocarpus foetidus, was discovered. The identified thermogenic oscillator had an accurate periodical cycle (ca. 60 min per cycle) that apparently responded to an increase or decrease in the spadix temperature with a threshold of less than 0.9 degrees C. Neither a constant ambient air temperature nor transient changes in the ambient air temperature within 10 min (19 degrees C --> 15 degrees C --> 19 degrees C) induced the temperature oscillation in the spadix. Moreover, the periodical cycles were independent of the weight of the spadix (2.5-9.2 g) and the amplitudes of the temperature oscillations were correlated with the magnitude of the changes in the spadix temperatures. These results imply that periodical temperature oscillations in the spadix of S. foetidus possess a quantitative regulatory process that involves a temperature sensation and subsequent heat production. Based on these results, we propose a time-dependent thermogenic oscillatory model that acts as a precise thermal regulator under dynamic environmental temperature changes.

The spadix of skunk cabbage, Symplocarpus foetidus, is capable of maintaining an internal temperature of around 20 degrees C even when the ambient temperature drops to around 0 degrees C. To determine the crucial structure that is required for detection of ambient temperature signals, detailed measurements of the temperatures of the spadix were made under field conditions. The spadix temperature was well regulated even when the spathe or the leaf of the plant was removed. Furthermore, maintenance of the temperature of the central stalk at either 10 or 20 degrees C had no effect on the thermoregulation when the ambient temperature increased from 10 to 25 degrees C or decreased from 20 to 8 degrees C. Therefore, it seemed that the heat production in the spadix required neither the spathe, the leaf, nor the central stalk for perception of the external temperature signals. Finally, analysis of sugar composition in xylem exudates showed that the concentrations of sucrose, glucose, and fructose, all of which are potential energy sources of thermogenesis, did not change significantly at different ambient temperatures. It is concluded that the spadix is a unique organ in which the perception of ambient temperature signals and heat production occurs in S. foetidus.

The effects of temperature on pollen germination and pollen tube growth rate were measured in vitro in thermogenic skunk cabbage, Symplocarpus renifolius Schott ex Tzvelev, and related to floral temperatures in the field. This species has physiologically thermoregulatory spadices that maintain temperatures near 23°C, even in sub-freezing air. Tests at 8, 13, 18, 23, 28 and 33°C showed sharp optima at 23°C for both variables, and practically no development at 8°C. Thermogenesis is therefore a requirement for fertilization in early spring. The narrow temperature tolerance is probably related to a long period of evolution in flowers that thermoregulate within a narrow range.

The cyanide-resistant alternative oxidase (AOX) is a homodimeric protein whose activity can be regulated by the oxidation/reduction state and by α-keto acids. To further clarify the role of AOX in the skunk cabbage, Symplocarpus renifolius, we have performed expression and functional analyses of the encoding gene. Among the various tissues in the skunk cabbage, SrAOX transcripts were found to be specifically expressed in the thermogenic spadix. Moreover, our data demonstrate that the SrAOX protein exists as a non-covalently associated dimer in the thermogenic spadix, and is more sensitive to pyruvate than to other carboxylic acids. Our results suggest that the pyruvate-mediated modification of SrAOX activity plays a significant role in thermoregulation in the skunk cabbage.

This paper presents a model of the thermoregulation system of the spadix of skunk cabbage Symplocarpus foetidus which regulates its internal temperature at around 20 degrees C during flowering even when the ambient air temperature drops below freezing. From the temperature responses of the spadix to changing ambient air temperature, we assumed that the thermoregulation system of the spadix is probably one of negative feedback control. The feedback signals are based on the rate of temperature change of the spadix over time. A signal is factored into the biochemical energy generator, and becomes biochemical energy, some of which becomes heat. Comparing our proposed model temperature responses and those of the living spadix, we found good agreement. In the process of engineering the model, the existence of two regulatory pathways in the thermoregulation system was simulated, and our proposed model appears to provide the necessary elements to explain the fundamental mechanism of the thermoregulation system of S. foetidus.

Brachypodium distachyon is an emerging model plant for studying biological phenomena in temperate grasses. Study of the growth scale is essential to analyse spatio-temporal changes in molecular factors throughout the life cycle. For sensitive and robust staging based on morphology in B. distachyon , we demonstrated the utility of the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemical industry) scale, which is comparable to the Zadoks scale conventionally used for Triticeae crops. We compared the chronological progression of B. distachyon accessions Bd21 and Bd3-1, in addition to the progression of Chinese Spring wheat. The comparison of growth stages illustrates the morphological similarities and differences in the timing of life cycle events. Furthermore, we compared metabolite accumulation patterns across different growth stages and across different stress conditions using a widely targeted metabolome analysis. Metabolic profiling determined commonalities and specificities in chemical properties that were dependent on organisms, growth stages and/or stress conditions. Most metabolites accumulated equivalently in B. distachyon and wheat. This qualitative similarity indicated the superiority of B. distachyon as a model for Triticeae crops. The growth scale of B. distachyon should provide a conceptual framework for comparative analysis and for knowledge integration between this model grass and crops in the Pooideae subfamily.

Two distinct mitochondrial energy dissipating systems, alternative oxidase (AOX) and uncoupling protein (UCP), have been implicated as crucial components of thermogenesis in plants and animals, respectively. To further clarify the physiological roles of AOX and UCP during homeothermic heat production in the thermogenic skunk cabbage (Symplocarpus renifolius), we identified the thermogenic cells and performed expression and functional analyses of these genes in this organism. Thermographic analysis combined with in situ hybridization revealed that the putative thermogenic cells surround the stamens in the florets of skunk cabbage and coexpress transcripts for SrAOX, encoding Symplocarpus AOX, and SrUCPb, encoding a novel UCP that lacks a fifth transmembrane segment. Mitochondria isolated from the thermogenic florets exhibited substantial linoleic acid (LA)-inducible uncoupling activities. Moreover, our results demonstrate that LA is capable of inhibiting the mitochondrial AOX pathway, whereas the proportion of pyruvate-stimulated AOX capacity was not significantly affected by LA. Intriguingly, the protein expression levels for SrAOX and SrUCPb were unaffected even when the ambient air temperatures increased from 10.3°C to 23.1°C or from 8.3°C to 24.9°C. Thus, our results suggest that functional coexpression of AOX and UCP underlies the molecular basis of heat production, and that posttranslational modifications of these proteins play a crucial role in regulating homeothermic heat production under conditions of natural ambient temperature fluctuations in skunk cabbage.