Microbial communities in plant roots are shaped by complex interbacterial interactions, yet how these interactions translate into plant fitness remains poorly understood. In this study, 127 bacterial isolates were obtained from barley (Hordeum vulgare L.) roots of two cultivars grown in a non-fertilized field, representing 45 genera and 72 species. Screening identified isolates with growth-promoting, growth-reducing, and neutral phenotypes. Co-inoculation experiments using synthetic communities (SynComs) demonstrated that growth-promoting isolates effectively cancelled the inhibitory effects of growth-reducing isolates on barley seedling growth. Mechanistic investigation revealed that growth-promoting isolates Variovorax sp. 14F-2.1 and Pseudomonas sp. 37A kill growth-reducing isolates Flavobacterium sp. 2D-1 through direct cell-to-cell contact. Deletion of the Type VI secretion system (T6SS) gene tssA in Variovorax sp. 14F-2.1 substantially reduced this activity, implicating T6SS as a key antagonistic mechanism. Phytohormone profiling revealed that growth-promoting and neutral isolates, but not growth-reducing isolates, produce cytokinins, and only Variovorax sp. 14F-2.1 could degrade IAA, suggesting a potential hormonal basis for differential growth effects. A two-year field microbiome study showed that fertilization regimen and seasonal sampling times were dominant drivers of rhizosphere community composition, while bacterial inoculation had limited and inconsistent effects on microbial diversity and plant growth under field conditions. These results demonstrate that interbacterial antagonism is a key determinant of community-level plant growth outcomes and highlight the complexity of translating laboratory inoculant effects to field settings.

Seed dormancy is essential to avoid pre-harvest sprouting (PHS), and germination is vital for agricultural production planning and malting. The malting barley genetic resource Chikukei 9713 is PHS tolerant but quickly breaks seed dormancy after harvest. We found that Chikukei 9713 and the barley cultivar Seijo 17 were PHS tolerant because exon-9 of the seed dormancy gene sd1 was of the dormant type. However, the rapidity of breaking seed dormancy depended on the threshing method. Both varieties germinated slowly after hand threshing, whereas Chikukei 9713, but not Seijo 17, germinated quickly after machine threshing. Using the 94 F2 individuals and 94 F3 lines populations derived from a cross between Chikukei 9713 and Seijo 17, we found a genetic region that controlled the effect of the threshing method on breaking seed dormancy (named bsd1) located on the short arm of chromosome 2H (23.4-24.9 Mbp) with GRAS-Di (Genotyping by Random Amplicon Sequencing-Direct) and CAPS markers. Further analysis revealed that bsd1 had a maximum LOD score of 21.4 and explained 65% of the variance. Our findings regarding this novel gene are an important genetic resource for stable malting barley production and sowing planning. Utilizing bsd1 and other seed dormancy genes will enable breeding of PHS-resistant barley cultivars that germinate quickly after machine harvesting.

Current advances in next-generation sequencing (NGS) technology and assembling programs permit construct chromosome-level genome assemblies in various plants. In contrast to resequencing, the genome sequences provide comprehensive annotation data useful for plant genetics and breeding. Herein, we constructed a reference-based genome assembly of winter barley (H. vulgare ssp. vulgare) cv. 'Hayakiso 2' using long and short read NGS data and barley reference genome sequences from 'Morex'. We constructed 'Hayakiso 2' genome sequences covering 4.3 Gbp with 55,477 genes. Comparative genomics revealed that 14,106 genes had orthologs to two barley data, wheat (A, B, and D homoeologs, respectively), and rice. From the gene ontology analysis, 2,494 orthologs against wheat and rice but not two barley contained agricultural important genes, such as 'response to biotic and abiotic stress' and 'metabolic process'. Phylogenetic analysis using 76 pangenome data indicated that 'Hayakiso 2' was clustered into Japanese-type genomes with unique alleles. 'Hayakiso 2' genome sequences showed known genes related to flowering and facilitated barley breeding through the development of various markers related to agronomically important alleles such as tolerance to various types of biotic and abiotic stress. Therefore, 'Hayakiso 2' genome sequences will be used for the further barley breeding.

Duganella sp. strains R1T, R57T, and R64T, isolated from barley roots in Japan, are Gram-stain-negative, motile, rod-shaped bacteria. Duganella species abundantly colonized barley roots. Strains R1T, R57T, and R64T were capable of growth at 4 °C, suggesting adaptation to colonize winter barley roots. Strains R57T and R64T formed purple colonies, indicating violacein production, while strain R1T did not. Based on 16S rRNA gene sequence similarities, strains R1T, R57T, and R64T were most closely related to D. violaceipulchra HSC-15S17T (99.10

Barley (Hordeum vulgare L.) growth is often inhibited under waterlogged fields. Under waterlogging, a higher C/N ratio in soil easily leads to low oxygen concentration and soil reduction. Although the tolerant varieties for waterlogging and soil reduction were previously reported, those root growth under hypoxia remains unknown. In this study, more than 90% of total leaf area in Deder 2, Hayakiso 2 and Akashinriki remained green even under waterlogged and reduced soil condition at seedling stage. The root elongation of Deder 2 and Hayakiso 2 was similar between aerobic and hypoxic conditions. In comparison, severe leaf necrosis was observed in Haruna Nijo and Houshun. Those root elongation was significantly inhibited under hypoxia, as compared with that under aerobic condition. In conclusion, it was considered that barley varieties tolerant to waterlogged reduced soil showed the better root growth in hypoxia in this study.

Barley (Hordeum vulgare ssp. vulgare) is one of the first crops to be domesticated and is adapted to a wide range of environments. Worldwide barley germplasm collections possess valuable allelic variations that could further improve barley productivity. Although barley genomics has offered a global picture of allelic variation among varieties and its association with various agronomic traits, polymorphisms from East Asian varieties remain scarce. In this study, we analyze exome polymorphisms in a panel of 274 barley varieties collected worldwide, including 137 varieties from East Asian countries and Ethiopia. We reveal the underlying population structure and conduct genome-wide association studies for 10 agronomic traits. Moreover, we examin genome-wide associations for traits related to grain size such as awn length and glume length. Our results demonstrate the value of diverse barley germplasm panels containing Eastern varieties, highlighting their distinct genomic signatures relative to Western subpopulations.

Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from japonica rice cultivars “Koshihikari” × “Khao Nam Jen”, each with different robustness of flowering time to environmental fluctuations. The difference of flowering times in 9 years’ field tests was large in “Khao Nam Jen” (36.7 days) but small in “Koshihikari” (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a “Khao Nam Jen” segment on chromosome 11 showed 28.0 days’ difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from “Khao Nam Jen” in the region around Hd16 on chromosome 3 showed 23.4 days” difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days’ difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with “Koshihikari”. Our results unravel the molecular control of flowering time under ambient temperature fluctuations.

Barley is the fourth most produced cereal crop in the world and one of the major dietary sources of cadmium (Cd), which poses serious threats to human health. Here, we identify a gene that encodes a P-type heavy metal ATPase 3 (HvHMA3) responsible for grain Cd accumulation in barley. HvHMA3 from the high Cd barley variety Haruna Nijo in Japan and the low Cd variety BCS318 in Afghanistan shared 97% identity at the amino acid level. In addition, the HvHMA3 from both varieties showed similar transport activity for Cd and the same subcellular localization at the tonoplast. However, the expression of HvHMA3 was double in BCS318 than in Haruna Nijo. A 3.3-kilobase Sukkula-like transposable element was found to be inserted upstream of the gene in the low Cd variety, which functioned as a promoter and enhanced the expression of HvHMA3 . Introgression of this insertion to an elite barley cultivar through backcrossing resulted in decreased Cd accumulation in the grain grown in Cd-contaminated soil without yield penalty. The decreased Cd accumulation resulting from the insertion was also found in some other barley landraces in the world. Our results indicate that insertion of the Sukkula-like transposable element plays an important role in upregulating HvHMA3 expression.

Aims Longer root hair length (RHL) increases root surface area, thereby contributing to increased phosphorous uptake. This study aimed to identify useful germplasms and quantitative trait loci (QTL) for RHL improvement in wheat. Methods The genetic variation in RHL was examined using hydroponic culture in 117 hexaploid wheat strains. Rhizosheath size and phosphorous uptake ability were also examined using high P-fixing soils with extremely low phosphorous availability (Andisols). QTL analysis was performed using backcross inbred lines from a cross between a Spanish spelt strain and a bread wheat variety. Results A wide range in genetic variation was found, especially in spelt wheat and landraces of bread wheat, whereas modern bread wheat varieties tended to show shorter RHL. Interestingly, remarkably longer RHL were observed in Spanish spelt strains than in strains from other countries. RHL exhibited a significant positive correlation with rhizosheath size and phosphorous uptake in soils. A QTL on chromosome 2A (QRhl.obu-2A) conferring longer RHL by the allele of a Spanish spelt strain was identified. It was co-located with TaRSL4-2AS, a key regulator of root hair elongation. Conclusions Spanish spelt wheat strains could be unique germplasms that provide a useful allele for increasing RHL to enhance phosphorous uptake ability.

In order to train the neural network for plant phenotyping, a sufficient amount of training data must be prepared, which requires time-consuming manual data annotation process that often becomes the limiting step. Here, we show that an instance segmentation neural network aimed to phenotype the barley seed morphology of various cultivars, can be sufficiently trained purely by a synthetically generated dataset. Our attempt is based on the concept of domain randomization, where a large amount of image is generated by randomly orienting the seed object to a virtual canvas. The trained model showed 96% recall and 95% average Precision against the real-world test dataset. We show that our approach is effective also for various crops including rice, lettuce, oat, and wheat. Constructing and utilizing such synthetic data can be a powerful method to alleviate human labor costs for deploying deep learning-based analysis in the agricultural domain.

Agronomically important traits often develop during the later stages of crop growth as consequences of various plant-environment interactions. Therefore, the temporal physiological states that change and accumulate during the crop's life course can significantly affect the eventual phenotypic differences in agronomic traits among crop varieties. Thus, to improve productivity, it is important to elucidate the associations between temporal physiological responses during the growth of different crop varieties and their agronomic traits. However, data representing the dynamics and diversity of physiological states in plants grown under field conditions are sparse. In this study, we quantified the endogenous levels of five phytohormones - auxin, cytokinins (CKs), ABA, jasmonate and salicylic acid - in the leaves of eight diverse barley (Hordeum vulgare) accessions grown under field conditions sampled weekly over their life course to assess the ongoing fluctuations in hormone levels in the different accessions under field growth conditions. Notably, we observed enormous changes over time in the development-related plant hormones, such as auxin and CKs. Using 3' RNA-seq-based transcriptome data from the same samples, we investigated the expression of barley genes orthologous to known hormone-related genes of Arabidopsis throughout the life course. These data illustrated the dynamics and diversity of the physiological states of these field-grown barley accessions. Together, our findings provide new insights into plant-environment interactions, highlighting that there is cultivar diversity in physiological responses during growth under field conditions.

Incorporating deep learning in the image analysis pipeline has opened the possibility of introducing precision phenotyping in the field of agriculture. However, to train the neural network, a sufficient amount of training data must be prepared, which requires a time-consuming manual data annotation process that often becomes the limiting step. Here, we show that an instance segmentation neural network (Mask R-CNN) aimed to phenotype the barley seed morphology of various cultivars, can be sufficiently trained purely by a synthetically generated dataset. Our attempt is based on the concept of , where a large amount of image is generated by randomly orienting the seed object to a virtual canvas. After training with such a dataset, performance based on recall and the average Precision of the real-world test dataset achieved 96% and 95%, respectively. Applying our pipeline enables extraction of morphological parameters at a large scale, enabling precise characterization of the natural variation of barley from a multivariate perspective. Importantly, we show that our approach is effective not only for barley seeds but also for various crops including rice, lettuce, oat, and wheat, and thus supporting the fact that the performance benefits of this technique is generic. We propose that constructing and utilizing such synthetic data can be a powerful method to alleviate human labor costs needed to prepare the training dataset for deep learning in the agricultural domain.

Aluminum (Al) toxicity is a major stress factor limiting crop productivity in acid soil. Although there is great genotypic variation in tolerance to Al toxicity, the underlying molecular mechanisms are poorly understood. Here, we report that, in barley (Hordeum vulgare), the fourth largest cereal crop produced in the world, both retrotransposon insertion and DNA methylation are involved in regulating differential Al tolerance. HvAACT1 is a major gene responsible for citrate secretion from the roots for external detoxification of Al. A multiretrotransposon-like (MRL) sequence insertion at least 15.3 kb in length was detected in the upstream genomic region of HvAACT1 that displayed promoter activity and significantly enhanced HvAACT1 expression, especially in the root tips of Al-tolerant accessions. Furthermore, in a number of accessions with low levels of HvAACT1 expression, this MRL insertion was present but highly methylated. Geographical analysis showed that accessions with this MRL insertion are distributed mainly in European areas with acid soils. Two wild barley accessions were found to possess this MRL insertion, but with a high degree of methylation. These results indicate that the MRL insertion and its degree of DNA methylation influence HvAACT1 expression and that demethylation of this MRL insertion, which facilitates adaptation to acid soils, occurred following barley domestication. Moreover, our results indicate that barley accessions in East Asia and Europe have developed independent but equivalent strategies to withstand Al toxicity in acid soils.

Adaptation to edaphic stress may have a key role in plant species range expansion. Aegilops tauschii Coss., the common wheat’s D-genome progenitor native to the Transcaucasus-Middle East region, is a good model to study the relationships between soil salinity and plant distributions: one of its intraspecific sublineages, TauL1b, drove the long-distance eastward expansion of this species range reaching semi-arid-central Asia. Salt tolerance during germination and seedling growth was evaluated in 206 Ae. tauschii accessions by treating seeds with NaCl solutions differing in concentrations. Differences in natural variation patterns were analyzed between sublineages and associated with natural edaphic condition variables, and then compared with reproductive trait variation patterns. The natural variations observed in NaCl-induced-stress tolerance had clear geographic and genetic structure. Seedling growth significantly increased in the TauL1b accessions that were collected from salt-affected soil habitats, whereas germinability did not. Principal component analysis suggested that the NaCl-induced-stress tolerances and reproductive traits might have had a similar degree of influence on Ae. tauschii’s eastward range expansion. Adaptation to salt-affected soils through increased seedling growth was an important factor for the species’ successful colonization of the semi-arid central Asian habitats. TauL1b accessions might provide useful genetic resources for salt-tolerant wheat breeds.

Barley is one of the founder crops of Old world agriculture and has become the fourth most important cereal worldwide. Information on genome-scale DNA polymorphisms allows elucidating the evolutionary history behind domestication, as well as discovering and isolating useful genes for molecular breeding. Deep transcriptome sequencing enables the exploration of sequence variations in transcribed sequences; such analysis is particularly useful for species with large and complex genomes, such as barley. In this study, we performed RNA sequencing of 20 barley accessions, comprising representatives of several biogeographic regions and a wild ancestor. We identified 38,729 to 79,949 SNPs in the 19 domesticated accessions and 55,403 SNPs in the wild barley and revealed their genome-wide distribution using a reference genome. Genome-scale comparisons among accessions showed a clear differentiation between oriental and occidental barley populations. The results based on population structure analyses provide genome-scale properties of sub-populations grouped to oriental, occidental and marginal groups in barley. Our findings suggest that the oriental population of domesticated barley has genomic variations distinct from those in occidental groups, which might have contributed to barley’s domestication.

Crops are exposed to various environmental stresses in the field throughout their life cycle. Modern plant science has provided remarkable insights into the molecular networks of plant stress responses in laboratory conditions, but the responses of different crops to environmental stresses in the field need to be elucidated. Recent advances in omics analytical techniques and information technology have enabled us to integrate data from a spectrum of physiological metrics of field crops. The interdisciplinary efforts of plant science and data science enable us to explore factors that affect crop productivity and identify stress tolerance-related genes and alleles. Here, we describe recent advances in technologies that are key components for data driven crop design, such as population genomics, chronological omics analyses, and computer-aided molecular network prediction. Integration of the outcomes from these technologies will accelerate our understanding of crop phenology under practical field situations and identify key characteristics to represent crop stress status. These elements would help us to genetically engineer "designed crops" to prevent yield shortfalls because of environmental fluctuations due to future climate change.

The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum vulgare ssp. vulgare) var. Hayakiso 2 (HK2) was developed by introducing VERNALIZATION-H1 (Vrn-H1) for spring growth habit from the spring-type var. Indo Omugi. Contrary to expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic difference was controlled by a single gene, which cosegregated only with phytochrome C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and CASEIN KINASE II alpha), indicating that HvPhyC was the most likely candidate gene. Compared with the late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a nonsynonymous amino acid substitution of phenylalanine at position 380 by serine in the functionally essential GAF (3',5'-cyclic-GMP phosphodiesterase, adenylate cyclase, formate hydrogen lyase activator protein) domain. Functional assay using a rice (Oryza sativa) phyA phyC double mutant line showed that both of the HvPhyC alleles are functional, but HvPhyC-e may have a hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL [HvPhyC-e] and NIL [HvPhyC-l], respectively) showed that HvPhyC-e up-regulated only the flowering promoter FLOWERING LOCUS T1 by bypassing the circadian clock genes and flowering integrator CONSTANS1 under a long photoperiod. Consistent with the up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long photoperiods. These results implied that HvPhyC is a key factor to control long-day flowering directly.

Originating from the Fertile Crescent in the Middle East, barley has now been cultivated widely on different soil types including acid soils, where aluminium toxicity is a major limiting factor. Here we show that the adaptation of barley to acid soils is achieved by the modification of a single gene (HvAACT1) encoding a citrate transporter. We find that the primary function of this protein is to release citrate from the root pericycle cells to the xylem to facilitate the translocation of iron from roots to shoots. However, a 1-kb insertion in the upstream of the HvAACT1 coding region occurring only in the Al-tolerant accessions, enhances its expression and alters the location of expression to the root tips. The altered HvAACT1 has an important role in detoxifying aluminium by secreting citrate to the rhizosphere. Thus, the insertion of a 1-kb sequence in the HvAACT1 upstream enables barley to adapt to acidic soils.