Supernumerary (B) chromosomes are widespread genomic elements that persist through non-Mendelian inheritance by exploiting host cellular mechanisms, yet the basis of their selective transmission and elimination remains poorly understood. This review integrates current knowledge on chromosome-specific behaviour, with particular emphasis on the centromere function, kinetochore assembly, epigenetic chromatin states, non-coding RNAs, and sequence composition. We examine how perturbations in these systems can skew chromosome segregation, or lead to chromosome elimination, and consider growing evidence that B chromosomes may themselves encode or modulate factors influencing these processes. Their repeat-rich architecture and enrichment in chromosome-specific satellite DNA are also discussed as contributors to their recognition by the cellular machinery. By unifying structural, epigenetic, and genetic perspectives, this review outlines a framework for understanding chromosome drive and elimination and highlights key directions for future research.

The correct tools for characterization of histone proteoforms are essential for deciphering plant epigenetic mechanisms and their subsequent application in biotechnology. Insights into the epigenetic landscape of plant chromatin can be advanced using bottom-up proteomics. MS analysis of histone peptides relies on careful sample preparation, including chemical derivatization of amine groups prior to MS to improve their chromatographic behaviour during nanoHPLC separation. Characterizing histones in plant tissues remains especially challenging due to the presence of diverse, species-specific compounds that interfere with MS analysis. In this study, we evaluated the impact of different protocols for the preparation of histones from maize (Zea mays) leaves on the quality of MS data. We were able to enhance the MS-based plant histone analysis protocol by combining chemical derivatization using trimethylacetic anhydride with enzymatic digestion with a novel protease, Arg-C Ultra. In addition, fluorescence-assisted cell sorting proved to be effective in isolating pure histone samples without the need for protein purification by precipitation. Our proposed new workflows produce highly pure histone extracts that are suitable for quantitative analysis of post-translational modifications and variant composition. They therefore offer a powerful tool for investigating epigenetic patterns and their dynamics in agriculturally important crops.

Abstract Programmed chromosome elimination is a highly controlled developmental process in which specific chromosomes are selectively lost from defined cell types during development. Despite its broad occurrence across plants and animals, the molecular mechanisms driving the tissue-specific elimination of chromosomes remain largely unresolved. Here, we exploit the root-specific elimination of supernumerary B chromosomes in Aegilops speltoides as a tractable model to identify the genetic basis of programmed chromosome loss. A high-quality, chromosome-scale genome assembly was generated, assigning 398 Mb of sequence to the Ae. speltoides B chromosome. Transcriptome profiling across seven tissue types representing chromosome elimination-active, elimination-negative, and B chromosome nondisjunction conditions identified 3,262 genes consistently upregulated in elimination-associated tissues, including 1,035 B genes. Stepwise subtraction of genes expressed in post-elimination and B chromosome-retaining reference tissues, followed by intersection with genes expressed during B nondisjunction in anthers, identified a candidate gene set enriched for chromosome segregation functions. From this set, we prioritized SYN2-B , a B chromosome-encoded cohesin α-kleisin subunit whose Arabidopsis thaliana ortholog AtSYN2 induces chromosome bridges and micronucleus formation when misregulated. CENH3-B , an α-type centromeric histone variant identified through GO enrichment analysis of B genes expressed in elimination-associated tissues, was shown to be incorporated in centromeres of both A and B chromosomes by transient gene expression assays using protoplasts and 3D structured illumination microscopy. These findings support a model in which B chromosome-encoded perturbations of cohesin activity and centromere composition contribute to selective B chromosome nondisjunction and their elimination in root tissues. Moreover, the SYN2-B promoter is enriched for ethylene response factor-binding sites compared to its A-encoded paralog, suggesting that ethylene is implicated in the root identity pathway driving root-specific B chromosome elimination.

Maize (Zea mays) is one of the world's most important crops and a recognized model for biological research, with some individuals having supernumerary B chromosomes. This B chromosome has been studied for decades, yet its gene expression across different plant tissues has not been thoroughly described. Here, we present a comprehensive transcriptomic atlas of the maize plant with and without the B chromosome. By analysing 11 tissues/organs, we found that genes encoded by the B chromosome contribute to the transcriptome throughout plant growth, with the highest activity observed in reproductive organs. Co-expression analysis revealed a cluster of 30 genes expressed specifically in tassels and indicated that Shortage in chiasmata 1 is a promising candidate for regulation of crossover frequency mediated by the B chromosome. In addition to its own transcriptional activity, our results also demonstrated that the B chromosome influences the expression of genes located on the A chromosome in all the tissues that we analysed. As well as providing new insights into the expression and regulatory effects of the B chromosome, our study has also generated fundamental information that will provide a basis for exploring its wider biological role.

Selective DNA elimination occurs across diverse species and plays a crucial role in evolution and development. This process encompasses small deletions, complete removal of chromosomes, or even the elimination of entire parental genomes. Despite its importance, the molecular mechanisms governing selective DNA elimination remain poorly understood. Our study focuses on the tissue-specific elimination of Sorghum purpureosericeum B chromosomes during embryo development. In situ B chromosome visualisation, complemented by transcriptomic profiling and gene-enrichment analysis, allows us to identify 28 candidate genes potentially linked to chromosome elimination. We show that elimination is a developmentally programmed process, peaking during mid-embryogenesis and nearly completed at later stages, leaving B chromosomes only in restricted meristematic regions. Genome sequencing reveals that the sorghum B chromosome is of multi-A chromosomal origin, has reduced gene density, is enriched in repetitive sequences, and carries a novel centromeric repeat, SpuCL166. Transcriptome analyses identify B-specific variants of kinetochore, cohesion, and checkpoint genes that are expressed during active elimination, while structural modeling of CENH3 and CENP-C indicates functional divergence at the kinetochore interface. Here, we provide the first comprehensive genomic and transcriptomic characterization of B chromosome and its elimination in Sorghum purpureosericeum. Our findings suggest that B chromosomes express modified mitotic machinery to control their own fate. By establishing a framework of candidate genes, this study opens new avenues for dissecting the molecular mechanisms of chromosome elimination and provides a critical foundation for understanding how genomes evolve to regulate and tolerate supernumerary chromosomal elements.

The B chromosomes exhibit diverse behaviour compared with conventional genetic models. The capacity of the B chromosome either to accumulate or to be eliminated in a tissue-specific manner is dependent on biological processes related to aberrant cell division(s), but here yet remains compatible with normal development. We studied B chromosome elimination in Sorghum purpureosericeum embryos through cryo-sections and demonstrated the B chromosome instability during plant growth using flow cytometry, molecular markers and fluorescent in situ hybridization techniques. Consequently, using B chromosome-specific probes we revealed the non-Mendelian inheritance of B chromosomes in developing pollen. We disclosed that the occurrence of the B chromosome is specific to certain tissues or organs. The distribution pattern is mainly caused by an extensive elimination that functions primarily during embryo development and persists throughout plant development. Furthermore, we described that B chromosome accumulation can occur either by nondisjunction at first pollen mitosis (PMI) or the initiation of extra nuclear division(s) during pollen development. Our study demonstrates the existence of a not-yet-fully described B chromosome drive process, which is likely under the control of the B chromosome.

Key message Characterisation and genetic mapping of a key gene defining root morphology in bread wheat. Abstract Root morphology is central to plants for the efficient uptake up of soil water and mineral nutrients. Here we describe a conditional mutant of hexaploid wheat ( Triticum aestivum L.) that when grown in soil with high Ca 2+ develops a larger rhizosheath accompanied with shorter roots than the wild type. In wheat, rhizosheath size is a reliable surrogate for root hair length and this was verified in the mutant which possessed longer root hairs than the wild type when grown in high Ca 2+ soil. We named the mutant Stumpy and showed it to be due to a single semi-dominant mutation. The short root phenotype at high Ca 2+ was due to reduced cellular elongation which might also explain the long root hair phenotype. Analysis of root cell walls showed that the polysaccharide composition of Stumpy roots is remodelled when grown at non-permissive (high) Ca 2+ concentrations. The mutation mapped to chromosome 7B and sequencing of the 7B chromosomes in both wild type and Stumpy identified a candidate gene underlying the Stumpy mutation. As part of the process to determine whether the candidate gene was causative, we identified wheat lines in a Cadenza TILLING population with large rhizosheaths but accompanied with normal root length. This finding illustrates the potential of manipulating the gene to disconnect root length from root hair length as a means of developing wheat lines with improved efficiency of nutrient and water uptake. The Stumpy mutant will be valuable for understanding the mechanisms that regulate root morphology in wheat.

The introgression of chromosome segments from wild relatives is an established strategy to enrich crop germplasm with disease-resistance genes 1 . Here we use mutagenesis and transcriptome sequencing to clone the leaf rust resistance gene Lr9 , which was introduced into bread wheat from the wild grass species Aegilops umbellulata 2 . We established that Lr9 encodes an unusual tandem kinase fusion protein. Long-read sequencing of a wheat Lr9 introgression line and the putative Ae. umbellulata Lr9 donor enabled us to assemble the ~28.4-Mb Lr9 translocation and to identify the translocation breakpoint. We likewise cloned Lr58 , which was reportedly introgressed from Aegilops triuncialis 3 , but has an identical coding sequence compared to Lr9 . Cytogenetic and haplotype analyses corroborate that the two genes originate from the same translocation event. Our work sheds light on the emerging role of kinase fusion proteins in wheat disease resistance, expanding the repertoire of disease-resistance genes for breeding.

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool 1 . Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43 , which was crossed into bread wheat from the wild grass Thinopyrum elongatum 2 , 3 . Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

BackgroundB chromosomes are classified as dispensable genomic components tolerated by cells, which are transmitted to progeny despite providing no benefit in most cases. They have been observed in over 2800 species of plants, animals and fungi, including numerous maize accessions. As maize is one of the most important crops worldwide, research on the maize B chromosome has been pioneering in the field. The characteristic of the B chromosome is its irregular inheritance. This results in offspring with a different number of B chromosomes compared to the parents. However, the exact number of B chromosomes in the studied plants is a crucial piece of information. Currently, assessing the number of B chromosomes in maize largely depends on cytogenetic analyses, which are laborious and time-consuming. We present an alternative approach based on the droplet digital PCR technique (ddPCR), which is faster, more efficient and provides the results within one day with the same level of accuracy.ResultsIn this study, we report a rapid and straightforward protocol for determining the number of B chromosomes in maize plants. We developed a droplet digital PCR assay using specific primers and a TaqMan probe for the B-chromosome-linked gene and a single-copy reference gene on maize chromosome 1. The performance of the assay was successfully verified by comparison with the results of cytogenetic analyses performed in parallel.ConclusionsThe protocol significantly improves the efficiency of B chromosome number assessment in maize compared to cytogenetic approaches. The assay has been developed to target conserved genomic regions and can therefore be applied to a wide range of diverged maize accessions. This universal approach can be modified for chromosome number detection in other species, not only for the B chromosome but also for any other chromosome in aneuploid constitution.

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

[目的]本文旨在开发纤毛鹅观草特异的寡核苷酸(oligonucleotide,oligo)探针,进一步完善纤毛鹅观草染色体鉴定技术.[方法]利用前期选育的普通小麦-纤毛鹅观草异附加系DA2Sc L和DA6Sc,通过流式分拣和基因组二代测序获得纤毛鹅观草染色体6Sc和染色体臂2Sc长臂的基因组序列,利用Repeatexplorer2/TAREAN软件从中鉴定出卫星重复序列并设计成oligo探针,通过荧光原位杂交(fluorescence in situ hybridization,FISH)分析所开发oligo探针的应用价值.[结果]从6Sc和2Sc L基因组序列中共鉴定出11个卫星重复序列并开发成11个oligo探针,oligo-FISH结果表明,其中6个探针可以在纤毛鹅观草染色体上产生明显杂交信号,而在小麦染色体上未产生明显杂交信号,可用于特异鉴定小麦背景中的纤毛鹅观草染色体或片段.对1套普通小麦-纤毛鹅观草异附加系oligo-FISH分析发现,oligo-2Sc L-163和oligo-6Sc-111仅在Sc基因组染色体上产生明显信号,可作为纤毛鹅观草Sc组特异探针;将oligo-2Sc L-161和oligo-2Sc L-163组合,对1整套二体异附加系进行双色oligo-FISH,构建了纤毛鹅观草染色体的oligo-FISH核型.[结论]本研究提供了纤毛鹅观草重复序列组成的初步信息,开发的探针能特异鉴定纤毛鹅观草染色体,阐明纤毛鹅观草2个基因组的来源和分化,对推动纤毛鹅观草优异基因的转移和利用有重要意义.

Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.

Chickpea (Cicer arietinum L.) is one of the main sources of plant proteins in the Indian subcontinent and West Asia, where two different morphotypes, desi and kabuli, are grown. Despite the progress in genome mapping and sequencing, the knowledge of the chickpea genome at the chromosomal level, including the long-range molecular chromosome organization, is limited. Earlier cytogenetic studies in chickpea suffered from a limited number of cytogenetic landmarks and did not permit to identify individual chromosomes in the metaphase spreads or to anchor pseudomolecules to chromosomes in situ. In this study, we developed a system for fast molecular karyotyping for both morphotypes of cultivated chickpea. We demonstrate that even draft genome sequences are adequate to develop oligo-fluorescence in situ hybridization (FISH) barcodes for the identification of chromosomes and comparative analysis among closely related chickpea genotypes. Our results show the potential of oligo-FISH barcoding for the identification of structural changes in chromosomes, which accompanied genome diversification among chickpea cultivars. Moreover, oligo-FISH barcoding in chickpea pointed out some problematic, most probably wrongly assembled regions of the pseudomolecules of both kabuli and desi reference genomes. Thus, oligo-FISH appears as a powerful tool not only for comparative karyotyping but also for the validation of genome assemblies.

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance ( R ) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62 , which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. We developed a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and used positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which was also transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines showed high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.