Due to large and complex genomes of Triticeae species, skim sequencing approaches have cost and analytical advantages for detecting genetic markers and building linkage maps. Here, we develop a ...high-density linkage map and identify quantitative trait loci (QTLs) for recombinant inbred lines of Aegilops tauschii, a D-genome donor of bread wheat, using the recently developed genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) system, which facilitates skimming of the large and complicated genome and generates a large number of genetic markers. The deduced linkage groups based on the GRAS-Di genetic markers corresponded to the chromosome number of Ae. tauschii. We successfully identified stable QTLs for flowering time and spikelet shape-related traits. Genotype differences of RILs at the QTL-linked markers were significantly associated with the trait variations. In particular, one of the QTL-linked markers for flowering time was mapped close to VRN3 (also known as FLOWERING LOCUS T), which controls flowering. The GRAS-Di system is, therefore, an efficient and useful application for genotyping and linkage mapping in species with large and complex genomes, such as Triticeae species.
RNA extraction has been improved by integration of a variety of materials in the protocol, such as phenol, guanidine thiocyanate, and silica, according to the case-specific demands. However, few ...methods have been designed for high-throughput RNA preparation for large-scale transcriptome studies. In this study, we established a high‐throughput guanidinium thiocyanate and isopropyl alcohol based RNA extraction method (HighGI). HighGI is based on simple and phenol-free homemade buffers and the cost is substantially lower than a column-based commercial kit. We demonstrated that the quality and quantity of RNA extracted with HighGI were comparable to those extracted with a conventional phenol/chloroform-based method and a column-based commercial kit. HighGI retained small RNAs less than 200 bp, which are lost with a commercial column-based kit. We also demonstrated that HighGI is readily applicable to semi-automated RNA extraction. HighGI enables high‐throughput RNA extraction for large-scale RNA preparation with high yield and quality.
Root system architecture affects the efficient uptake of water and nutrients in plants. The root growth angle, which is a critical component in determining root system architecture, is affected by ...root gravitropism; however, the mechanism of root gravitropism in rice remains largely unknown. In this study, we conducted a time-course transcriptome analysis of rice roots under conditions of simulated microgravity using a three-dimensional clinostat and following gravistimulation to detect candidate genes associated with the gravitropic response. We found that
(
) genes, which are involved in the regulation of auxin transport, were preferentially up-regulated during simulated microgravity conditions and rapidly down-regulated by gravistimulation. We also found that the transcription factor
A2s (
s) and
s, showed the similar expression patterns with the
s. A co-expression network analysis and an in silico motif search within the upstream regions of the co-expressed genes revealed possible transcriptional control of
s by HSFs. Because HSFA2s are transcriptional activators, whereas HSFB2s are transcriptional repressors, the results suggest that the gene regulatory networks governed by HSFs modulate the gravitropic response through transcriptional control of
s in rice roots.
SUMMARY
A cultivation facility that can assist users in controlling the soil water condition is needed for accurately phenotyping plants under drought stress in an artificial environment. Here we ...report the Internet of Things‐based pot system controlling optional treatment of soil water condition (iPOTs), an automatic irrigation system that mimics the drought condition in a growth chamber. The Wi‐Fi‐enabled iPOTs system allows water supply from the bottom of the pot, based on the soil water level set by the user, and automatically controls the soil water level at a desired depth. The iPOTs also allows users to monitor environmental parameters, such as soil temperature, air temperature, humidity, and light intensity, in each pot. To verify whether the iPOTs mimics the drought condition, we conducted a drought stress test on rice (Oryza sativa L.) varieties and near‐isogenic lines, with diverse root system architecture, using the iPOTs system installed in a growth chamber. Similar to the results of a previous drought stress field trial, the growth of shallow‐rooted rice accessions was severely affected by drought stress compared with that of deep‐rooted accessions. The microclimate data obtained using the iPOTs system increased the accuracy of plant growth evaluation. Transcriptome analysis revealed that pot positions in the growth chamber had little impact on plant growth. Together, these results suggest that the iPOTs system is a reliable platform for phenotyping plants under drought stress.
Significance statement
We developed the Internet of Things‐based pot system controlling optional treatment of soil water condition (iPOTs), an automatic irrigation system to mimic the drought condition, for phenotyping plants in a growth chamber. Bottom irrigation allows the user to adjust the soil water level. Aerial and underground sensors in each pot enable the assessment of the effect of microclimate on plant growth. Phenome and transcriptome analyses demonstrated that the iPOTs system is a reliable platform for plant phenotyping under artificial drought stress.
The popular ornamental flowering plant Dianthus hybrida cv. Telstar Scarlet has been found to exhibit two populations, each with distinct flower morphology: female-like and hermaphroditic. In this ...work, flower development of D. hybrida was characterized through scanning electron microscopy, light- or stereo- microscopy, from flower meristem formation to the fully matured, open flower. The difference between hermaphrodite and female-like plants was initially marked by the phenomenon of anther shrinking in the latter, which was closely associated with pollen shrinking phenomenon, and soon followed by differential elongation rates of pistils and stamens. Furthermore, the female-like anther, albeit exhibiting delay in pollen development initially, could produce microspores, resembling its hermaphrodite counterparts at some point, before shrinking. However, female-like’s pollen and cell wall size never became as large as the hermaphrodite’s.
Wild diploid wheat Aegilops tauschii, the D-genome progenitor of common wheat, carries large genetic variation in spikelet and grain morphology. Two differentiated subspecies of Ae. tauschii, ...subspecies tauschii and strangulata, have been traditionally defined based on differences in spikelet morphology. Here, we first assessed six spikelet shape-related traits among 199 Ae. tauschii accessions, and found that the accessions belonging to TauL1major lineage produced significantly longer spikes, higher spikelet density, and shorter, narrower spikelets than another major lineage, TauL2, in which the strangulata accessions are included. Next, we performed quantitative trait locus (QTL) analysis of the spikelet and grain shape using three mapping populations derived from interlineage crosses between TauL1 and TauL2 to identify the genetic loci for the morphological variations of the spikelet and grain shape in Ae. tauschii. Three major QTL regions for the examined traits were detected on chromosomes 3D, 4D and 7D. The 3D and 4D QTL regions for several spikelet shape-related traits were conserved in the three mapping populations, which indicated that the 3D and 4D QTLs contribute to divergence of the two major lineages. The 7D QTLs were found only in a mapping population from a cross of the two subspecies, suggesting that these 7D QTLs may be closely related to subspecies differentiation in Ae. tauschii. Thus, QTL analysis for spikelet and grain morphology may provide useful information to elucidate the evolutionary processes of intraspecific differentiation.
Touch stimulus responses are common in plants. Some flowering plants sense the arrival of their pollinators and secrete nectar or release pollen sacs, facilitating successful pollination. Molecular ...mechanisms for mechanical stimulus responses in plants are well characterized in Arabidopsis leaves, but not in non-model plants or other organs such as flowers. Here, we performed RNA-seq analysis of touched flower buds of Dianthus hybrida, a major ornamental plant. Upon touch treatment, 931 and 132 genes were upregulated and downregulated, respectively. GO enrichment analysis revealed that genes encoding serine/threonine protein kinases were significantly abundant among the upregulated genes, which is consistent with previous studies that demonstrated the pivotal role of protein phosphorylation in the touch stimulus response of Arabidopsis leaves. In comparison with the gene expression profile of touched Arabidopsis leaves, the same families but different homologs of the representative touch-induced genes encoding protein kinases were upregulated, showing that phosphorelay signaling was the common mechanism for touch stimulus response in flowers and leaves, but the players of the phosphorelay signaling were different. These results will contribute to further studies on the mechanical stimulus responses of ornamental flowers and the utilization of this mechanism for breeding programs.
Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, has been the source of trait improvement in wheat breeding. Intraspecific genetic variation of Ae. umbellulata, however, has not ...been well studied and the genomic information in this species is limited.
To develop novel genetic markers distributed over all chromosomes of Ae. umbellulata and to evaluate its genetic diversity, we performed RNA sequencing of 12 representative accessions and reconstructed transcripts by de novo assembly of reads for each accession. A large number of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were obtained and anchored to the pseudomolecules of Ae. tauschii and barley (Hordeum vulgare L.), which were regarded as virtual chromosomes of Ae. umbellulata. Interestingly, genetic diversity in Ae. umbellulata was higher than in Ae. tauschii, despite the narrow habitat of Ae. umbellulata. Comparative analyses of nucleotide polymorphisms between Ae. umbellulata and Ae. tauschii revealed no clear lineage differentiation and existence of alleles with rarer frequencies predominantly in Ae. umbellulata, with patterns clearly distinct from those in Ae. tauschii.
The anchored SNPs, covering all chromosomes, provide sufficient genetic markers between Ae. umbellulata accessions. The alleles with rarer frequencies might be the main source of the high genetic diversity in Ae. umbellulata.
The wild species in the Triticeae tribe are tremendous resources for crop breeding due to their abundant natural variation. However, their huge and highly repetitive genomes have hindered the ...establishment of physical maps and the completeness of their genome sequences. To develop molecular markers for the efficient utilization of their valuable traits while avoiding their genome complexity, we assembled RNA sequences of ten representative accessions of
Aegilops tauschii
, the progenitor of the wheat D genome, and estimated single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). The deduced unigenes were anchored to the chromosomes of
Ae. tauschii
and barley. The SNPs and indels in the anchored unigenes, covering entire chromosomes, were sufficient for linkage map construction, even in combinations between the genetically closest accessions. Interestingly, the resolution of SNP and indel distribution on barley chromosomes was slightly higher than on
Ae. tauschii
chromosomes. Since barley chromosomes are regarded as virtual chromosomes of Triticeae species, our strategy allows capture of genetic markers arranged on the chromosomes in order based on the conserved synteny. The resolution of these genetic markers will be comparable to that of the
Ae. tauschii
whose draft genome sequence is available. Our procedure should be applicable to marker development for Triticeae species, which have no draft sequences available.
Cytology requires chromosome specimens, thus, effective preparation methods are needed. Chromosome specimens are frequently prepared from plant root tips. Furthermore, cell cycle synchronization ...using chemical reagents is applied to obtain a large number of metaphase chromosome specimens. In this study, we focused on the timing of root tip sampling, which is optimal for the preparation of chromosome specimens of the dioecious plant Silene latifolia. The timing was determined from the time seeds were subjected to a germination treatment. Observation of metaphase chromosomes using microscopy revealed that the number of mitotic cells peaked 54 h after the germination treatment. This trend was also observed when the DNA synthesis inhibitor aphidicolin was administered from 24 to 9 h before sampling time points. We used ice-cold treatment for 8, 16, and 32 h as a chromosome condensation method. The 16 h treatment produced suitable chromosome specimens showing satellite ends of chromosomes, whereas the 32 h treatment produced well-condensed chromosome specimens, which were suitable for counting chromosome numbers. Our findings suggest that the timing of root tip sampling is essential for effectively producing plant chromosome specimens.
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