Shun Sakuma

The genus Aegilops comprises important wild relatives of cultivated wheat and represents a valuable genetic resource for wheat improvement. In this study, the complete chloroplast genomes of five Aegilops species (Ae. crassa, Ae. cylindrica, Ae. juvenalis, Ae. tauschii, and Ae. triuncialis) collected from Kazakhstan and Uzbekistan were sequenced, assembled, and comparatively analyzed. The chloroplast genomes exhibited a conserved quadripartite structure consisting of a large single-copy (LSC), a small single-copy (SSC), and two inverted repeat (IR) regions. Genome sizes ranged from 135,612 to 136,840 bp, with an identical GC content of 38% across all species. Comparative analyses revealed high structural conservation among chloroplast genomes, particularly within IR regions, whereas greater sequence divergence was observed in the non-coding regions of the LSC and SSC. Sliding-window analysis identified several highly polymorphic regions, including rpl32-trnL(UAG), ndhF-rpl32, trnC(GCA)-rpoA, psbA, and ndhD, which may serve as potential DNA barcodes and informative markers for phylogenetic studies. A total of 850 chloroplast simple sequence repeats (SSRs) were detected, predominantly A/T-rich mononucleotide repeats. Codon usage analysis demonstrated a conserved preference for A/U-ending codons across all species. Ka/Ks analysis indicated that most chloroplast protein-coding genes are under strong purifying selection, although relatively elevated evolutionary rates were detected in rpoA and ycf4. Phylogenetic analyses based on complete chloroplast genomes strongly supported sectional relationships within Aegilops and confirmed close maternal relationships among several species. Overall, this study provides chloroplast genome resources for Aegilops and contributes to understanding chloroplast genome evolution, phylogeny, and molecular marker development.

Grain yield in wheat is frequently driven by pre-anthesis growth and how carbon is directed to reproductive parts, determining the potential size of the grain-producing sink. However, the genetic mechanisms controlling this carbon allocation remain unclear. In this study we discovered a series of loci in tetraploid and hexaploid wheats that, when combined, confer an average grain yield advantage in hexaploid winter wheat of approx. 5-7% under high-input farming while approx. 10-15% under low-input conditions. The responsible gene is GRAIN NUMBER INCREASE 2 ( GNI2 ), ancestral to its duplicate GNI1 , and represented by the homoeo-allelic series GNI-A2 , GNI-B2 , and GNI-D2 . GNI1 and all GNI2 copies additively affect floral growth and fertility by modulating reproductive allocation and harvest index. Herewith, we describe a series of grain yield-relevant GNI2 homoeo-alleles with a proven track record for being beneficial in both high- and low-input environments. Their deployment offers a sustainable pathway to raise global grain yields in the future.

Bread wheat ( Triticum aestivum L.) is one of the most important staple crops globally. Grain number per spikelet (GPS) is an important yield component in wheat. It is influenced by floret number per spikelet (FPS) and floret fertility. Through three consecutive years of observation of FPS, GPS, and floret fertility per spikelet (FFPS) in a globally collected hexaploid wheat core collection and using an improved genotyping dataset, we identified 42 marker‐trait associations (MTA) for spikelet‐related traits through the single‐nucleotide polymorphism and the k‐mer‐based genome‐wide association studies. Those MTA explain 4.45%–24.45% phenotypic variations for the FFPS and GPS traits, indicating a substantial effect on spikelet‐related traits. We found significant negative associations between days to heading (DTH) and spikelet‐related traits, suggesting that DTH and spikelet‐related traits are subject to a certain degree of common genetic control. We identified several potential candidate gene models in the associated regions, including TraesCS2A03G0815500, which encodes a protein with a sterile alpha motif domain, and TraesCS2A03G0780600, which encodes a plastid‐targeted lipoxygenase. These genes may facilitate wheat breeding and help elucidate the mechanisms of wheat grain set.

The genus Hordeum, which includes cultivated barley and its wild relatives, represents a diverse group of grasses with agricultural, ecological, and evolutionary importance. Here, we present a genus-wide pangenome based on 25 chromosome-scale genome sequences encompassing all known diploid Hordeum species and selected tetraploids. Comparative analysis reveals extensive structural variations, including large-scale inversions and chromosomal rearrangements, which disrupt proximal chromosomal collinearity and reflect lineage-specific genome remodeling. Using single-copy gene phylogenies, we identify multiple deep interspecific hybridization events, segmental exchanges, and introgressions, particularly in regions enriched for conserved inversions. We also uncover widespread horizontal gene transfer from Panicoideae grasses into Hordeum, with functional integration of transferred genes. Transposable element dynamics, especially retrotransposon proliferation, account for much of the observed genome size variation and offer insights into polyploid genome evolution. Whole-genome alignments reveal contrasting evolutionary patterns: while coding genes remain highly conserved, regulatory elements show rapid turnover, providing a substrate for phenotypic diversification. Case studies of vernalization and glume development demonstrate how natural variation in regulatory and coding sequences contributes to flowering time and floral morphology. This comprehensive pangenomic framework establishes Hordeum as a model for studying genome evolution, structural variation, and the genetic basis of adaptive traits, offering new avenues for barley improvement.

Successful pollination and fertilization are crucial for grain setting in cereals. Wheat is an allohexaploid autogamous species. Due to its evolutionary history, the genetic diversity of current bread wheat (Triticum aestivum) cultivars is limited. Introducing favorable alleles from related wild and cultivated wheat species is a promising breeding strategy for resolving this issue. However, wide hybridization between bread wheat and its relatives is hampered by the presence of suppressor genes and difficulties in crossing. Optimized methods for observing pollen tubes are essential for understanding the mechanism of crossability between wheat and its relatives. Here, we improved the crossing procedure between bread wheat and rye (Secale cereale) and established an optimized protocol for visualizing pollen tube behavior. Crossing via detached spike culture significantly enhanced crossing efficiency and phenotypic stability. A combination of canonical aniline blue staining and optimized clearing and sectioning allowed us to visualize pollen tube behavior. The proportion of rye pollen tubes reaching the micropyle was lower than that for pollen tubes germinated on the stigmatic hair, explaining why the hybrid seed-setting rate was approximately 75% instead of 100%. This method sheds light on wide hybridization through deeper visualization of the insides of pistils.

Pangenomes are collections of annotated genome sequences of multiple individuals of a species¹. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants². Here we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that are rich in gene copy number variation. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that allelic diversity at structurally complex loci may have helped crop plants to adapt to new selective regimes in agricultural ecosystems.

Pangenomes are collections of annotated genome sequences of multiple individuals of a species. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants. Here, we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that have become hot spots of gene copy number variation in evolutionarily recent times. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release, and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that rapid evolution at structurally complex loci may have helped crop plants adapt to new selective regimes in agricultural ecosystems.

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Modifying inflorescence architecture improves grain number and grain weight in bread wheat (Triticum aestivum). Allelic variation in Grain Number Increase 1 (GNI-A1) genes, encoding a homeodomain leucine zipper class I transcription factor, influences grain number and yield. However, allelic information about GNI-A1 in diverse germplasms remains limited. Here, we investigated GNI-A1 alleles in a panel of 252 diverse bread wheat accessions (NBRP core collection and HRO breeder’s panel) by target resequencing. Cultivars carrying the reduced-function allele (105Y) were predominant in the NBRP panel, whereas the 105N functional allele was the major type in the HRO panel. Cultivars with the 105Y allele were distributed in Asian landraces but not in European genotypes. Association analysis demonstrated that floret fertility, together with grain size, were improved in cultivars in the NBRP core collection carrying the 105Y allele. These results imply that different alleles of GNI-A1 have been locally selected, with the 105Y allele selected in East Asia and the 105N allele selected in Europe.