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.

Understanding relationships among pear (Pyrus spp.) accessions and ensuring their correct identification is critical for breeding and germplasm management. In this study, we analyzed 445 accessions, primarily Pyrus communis, using three genotyping approaches to assess population structure, determine parentage, and identify cultivars. ddRAD libraries were prepared using the restriction enzymes AvaII and MspI. From more than 7,000 SNPs pruned for linkage disequilibrium, we distinguished species, identified clones, commonly used breeding cultivars and their offspring, and detected misclassified accessions. From the identified SNPs, we developed a panel of over 100 amplicon-based SNP (abSNP) markers. In parallel, we designed a novel set of 17 SSR markers, allowing both marker types to be genotyped in a single PCR reaction and directly compared. The SSR panel proved highly robust, achieving a probability of identity (PID) of 9.1 & times; 10-25, which allowed for discrimination among individual accessions and facilitated parentage assignment. In contrast, abSNP markers were less reliable for parentage analysis due to amplification bias associated with the highly heterogeneous pear genome. Nevertheless, abSNP markers were highly effective for clone identification, cultivar discrimination, and population-level studies. These results provide a frameworkfor cost-effective genotyping and germplasm management in pear breeding programs.

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.

Sweet cherry (Prunus avium L.) exhibits relatively low genetic diversity because of the self-compatibility of some varieties and repeated crossings of the same genotypes. High-quality markers are therefore needed for their reliable discrimination. However, the most currently used simple sequence repeat (SSR) markers offer only limited resolution for genotyping purposes. Here, thirty new highly polymorphic SSR markers were extracted from whole-genome sequences of 299 sweet cherry genotypes. Then, 16 highly polymorphic SSR markers were selected, multiplexed into one PCR, and successfully verified on a collection containing 294 unique genotypes. Compared with the set of 16 SSR markers recommended by the European Cooperative Programme for Plant Genetic Resources (ECPGR) for sweet cherry genotyping, our newly developed system has a seven orders of magnitude lower probability of the random identity of two genetically distinct samples than the ECPGR set (10-19 vs. 10-12). This higher resolution not only enables more precise genotyping but can also be successfully used for parentage or population analyses. This new and unique one-tube approach for sweet cherry genotyping will substantially simplify genotyping workflows, minimize errors, and save labor, time, and cost.

In addition to wild relatives, cultivated Triticeae species such as barley can also serve as an excellent gene source to improve the agronomic traits of bread wheat. The Asakaze-Manas addition lines previously developed in Martonvásár exhibit low fertility and unstable inheritance; nonetheless, they could serve as ’bridge materials’ for the incorporation of barley chromatin into the wheat genome. By leveraging the centric breakage-fusion mechanism of unpaired chromosomes and the gametocidal effect of chromosome 2C from Aegilops cylindrica, the 4H and 6H addition lines were used to develop genetically stable translocation genotypes. In situ hybridization and molecular marker analyses were applied to identify the wheat and barley chromosome arms in the compensatory translocations, designated as T6HS.6BL, T6BS.6HL and T4BS.4HL. These results were confirmed by GBS read coverage analysis, which revealed a putative pericentric inversion in the barley chromosome arms involved in the T6HS.6BL and T6BS.6HL. The comparable agronomic performance of the centric fusion lines indicates that these chromosomal rearrangements had minimal, if any, negative impact relative to the parental wheat varieties. The newly developed translocation lines had an increased amount of minerals such as iron, suggesting that they could serve as promising genetic material for the biofortification of hexaploid wheat. Molecular marker analysis and in situ hybridization techniques, combined with genotyping-by-sequencing, enable the selection of stable wheat-barley translocation lines, leading to chromosome-mediated improvement in grain quality.

Triticum timopheevii (2n = 4x = 28, GGAtAt) is a valuable wild wheat relative for improving disease resistance and agronomic performance in bread wheat, although the detection of introgressed chromosomal segments remains challenging due to close genomic similarity. A wheat × T. timopheevii pre-breeding population was analyzed using Genotyping-by-sequencing (GBS) combined with a skim-seq pipeline to identify and characterize T. timopheevii introgressions. Read coverage analysis based on a combined T. aestivum–T. timopheevii reference genome enabled high-resolution detection of major chromosomal introgressions and copy-number changes. Multiple large-scale chromosomal rearrangements were identified, most frequently involving wheat chromosome 2B and T. timopheevii chromosome 2G, involving the replacement of a ~625 Mb wheat segment with a ~526 Mb T. timopheevii segment. Additional rearrangements included a 6A–6At translocation, a nullisomic 6B genotype, and widespread 1B.1R rye introgressions. These findings were confirmed by fluorescence in situ hybridization. Phenotyping of plant-pathogen interactions showed that some lines carrying 2G introgressions displayed reduced disease symptoms against individual leaf or yellow rust isolates. Several genotypes also showed favorable yield-component traits, including increased spikelet and grain number. Our results demonstrate that GBS-based read coverage analysis combined with cytogenetic validation is an effective approach for dissecting genome structure and identifying candidate introgressions with potential breeding relevance. The recurrent involvement of specific chromosomes highlights preferential homoeologous recombination patterns and supports the targeted utilization of T. timopheevii genetic diversity for wheat improvement.

Goatgrasses with U- and M-genomes are important sources of new alleles for wheat breeding to maintain yield and quality under extreme conditions. However, the introgression of beneficial traits from wild Aegilops species into wheat has been limited by poor knowledge of their genomes and scarcity of molecular tools. Here, we present the first linkage map of allotetraploid Aegilops biuncialis Vis., developed using 224 F2 individuals derived from a cross between MvGB382 and MvGB642 accessions. The map comprises 5663 DArTseq markers assigned to 15 linkage groups corresponding to 13 chromosomes. Chromosome 1Mb could not be constructed due to a lack of recombination caused by rearrangements in the MvGB382 accession. The genetic map spans 2518 cM with an average marker density of 2.79 cM. The skeleton map contains 920 segregating markers, divided between the Mb sub-genome (425 markers) and the Ub sub-genome (495 markers). Chromosomes of the Mb sub-genome, originating from Aegilops comosa Sm. in Sibth. et Sm., show well-preserved collinearity with Triticum aestivum L. chromosomes. In contrast, chromosomes of the Ub sub-genome, originating from Aegilops umbellulata Zhuk., exhibit a varying degree of collinearity, with 1Ub, 3Ub, and 5Ub retaining a substantial level of collinearity with Triticum aestivum, while 2Ub, 4Ub, 6Ub, and 7Ub show significant rearrangements. A quantitative trait locus affecting fertility was identified near the centromere on the long arm of chromosome 3Mb, explaining 23.5% of the variance. The genome structure of Aegilops biuncialis, highlighted by the genetic map, provides insights into the speciation within the species and will support alien gene transfer into wheat.

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.

The stable 3St(3D) substitution line offers promising genetic potential for improving drought tolerance in wheat during critical reproductive stages. The flowering stage is highly susceptible to drought, which significantly reduces wheat grain yield globally. Low genetic diversity in wheat further limits the discovery of optimal gene variants for breeding climate-resilient varieties. The substitution of chromosome 3D by a group 3 chromosome pair from Thinopyrum intermedium × Th. ponticum artificial hybrid was identified using in situ hybridization and genotyping-by-sequencing. This homoeologous substitution showed good functional compensation for grain yield and fertility, similar to the wheat parents ('Mv9kr1' and 'Mv Karizma') in field and greenhouse trials. The substitution line exhibits a semidwarf phenotype due to the Rht8 and Rht2 dwarfing alleles. Automated shoot phenotyping after a 10-day water withdrawal at flowering revealed efficient water preservation allowing to maintain photosynthetic functions, sustained photosynthetic activity, and less chlorophyll degradation, indicated by Normalized Difference Vegetation Index (NDVI) and modified Normalized Difference Index (mND705) values and moderate level of protective functions shown by the expression of stress-related genes. Compared to the wheat parents, the substitution line developed thicker roots with increased volume under drought, resulting in a lower surface-to-volume ratio. This may enhance water storage efficiency and help reduce yield loss under drought conditions.

Wheat and its close relatives have large and complex genomes, making gene cloning difficult. Nevertheless, developments in genomics over the past decade have made it more feasible. The large and complex genomes of cereals, especially bread wheat, have always been a challenge for gene mapping and cloning. Nevertheless, recent advances in genomics have led to significant progress in this field. Currently, high-quality reference sequences are available for major wheat species and their relatives. New high-throughput genotyping platforms and next-generation sequencing technologies combined with genome complexity reduction techniques and mutagenesis have opened new avenues for gene cloning. In this review, we provide a comprehensive overview of the genes cloned in wheat so far and discuss the strategies used for cloning these genes. We highlight the advantages and drawbacks of individual approaches and show how particular genomic progress contributed to wheat gene cloning. A wide range of new resources and approaches have led to a significant increase in the number of successful cloning projects over the past decade, demonstrating that it is now feasible to perform rapid gene cloning of agronomically important genes, even in a genome as large and complex as that of wheat.

Bread wheat, a global staple food, has seen a significant reduction in genetic diversity due to domestication and intensive breeding. Diploid einkorn wheat (Triticum monococcum L.) is a valuable resource for enhancing the genetic pool of bread wheat. A linkage map spanning 1033 cM was constructed using 81 recombinant inbred lines (RILs) derived from a cross between cultivated Triticum monococcum 'DV92' and wild Triticum boeoticum 'G3116.' The RILs were phenotyped in up to eight environments for 24 agronomically important traits, including plant height, tiller number, plant architecture, leaf pubescence, ear emergence time, and spike- and grain-related traits. QTL analyses revealed 167 quantitative trait loci (QTLs) distributed across all chromosomes, of which 36 QTLs were mapped in intervals of less than 10 cM, and 77 QTLs explained more than 10% of the phenotypic variance for a trait, with a maximum of 56.2% (the drought resistance-related leaf pubescence). Verification using 407 F2 genotypes confirmed that over 30% of the QTLs were highly reproducible. In addition, we have demonstrated that the identified QTLs can be readily characterized by fine-mapping of a novel QTL for leaf pubescence, QLP.ieb-3A, on chromosome 3Am. This study provides valuable insights into loci influencing key agronomic traits, such as yield and resistance to abiotic stress, and is suitable for bread wheat gene pool enrichment. Additionally, the findings facilitate map-based cloning of the underlying genes, paving the way for functional studies and efficient application of the loci in wheat breeding programs.

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.

Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.

Light is an essential environmental signal for plant development called photomorphogenesis. Here, we show that diethyl ether anaesthesia inhibits the de-etiolation process in barley (Hordeum vulgare) seedlings. Illuminated seedlings exposed to diethyl ether accumulated significantly less chlorophylls and chlorophyll-binding proteins, and exhibited reduced maximum quantum yield of photosystem II photochemistry (Fv/Fm). Although the direct effect of light necessary for the greening process, i.e. for the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) catalysed by light-dependent protochlorophyllide oxidoreductase A (PORA), was not inhibited, the RNA-seq and qPCR analyses showed that light-induced expression of photosynthesis-associated nuclear genes (PhANGs) and genes encoding enzymes for chlorophyll biosynthesis were attenuated. On the other hand, transcription of chloroplast-encoded genes was not negatively affected by diethyl ether treatment during greening. Among the genes negatively regulated by light, PORA and PHYA were only slightly affected by diethyl ether. The effect of diethyl ether was fully reversible and, after its removal, the greening process was fully restored. Our data indicate that diethyl ether had two effects on greening: i) it inhibited the expression of PhANGs and chlorophyll biosynthesis-related genes irrespective of light conditions, ii) it blocked the light-induced expression of these genes and greening process of etiolated seedlings. Our study indicates that diethyl ether affects plastid biogenesis, which alters the orchestration of negative and positive regulators affecting phytochrome and/or retrograde signalling and does not allow expression of PhANGs. Thus, the plants are locked in an intermediate skoto-photomorphogenetic state in the light.

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.

AbstractThe genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

SUMMARY Restoring cytonuclear stoichiometry is necessary after whole‐genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto‐ and allopolyploids of the Festuca‐Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well‐established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post‐transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

Sweet cherry (Prunus avium L.) is a fruit tree in the Rosaceae family grown worldwide for its tasty fruit. However, its yield may be threatened in warmer growing regions by insufficient dormancy, which usually occurs in late-blooming genotypes. Conversely, in cold regions, the yield is threatened by late spring frosts, especially for early flowering cultivars. It is therefore necessary to breed cultivars adapted to local weather conditions and avoid potential crop losses. New markers associated with the beginning of flowering were sought to enable molecular marker-assisted selection of genotypes tailored for different climatic conditions. Previously whole-genome sequenced 298 sweet cherry genotypes with nine years of phenotypic evaluation provided the basis for a genome-wide association study that allowed the identification of 163 single nucleotide polymorphisms and indels associated with flowering time, located on all sweet cherry chromosomes. This study confirmed the previously predicted polygenic basis of the trait. Three markers suitable for selection of late-blooming genotypes and one for early-blooming genotypes were selected and validated using independent 128 sweet cherry hybrids from different crossings. Individual markers for late beginning of flowering were able to select genotypes flowering at least three days after the reference (i.e. the earliest flowering) cultivar 'Kišiněvskaja'. Accumulation of preferred allele combinations for all three late-blooming markers has a synergistic effect, indicating delay of flowering 7.1 days after the reference cultivar on average. The marker for early beginning of flowering identified accessions flowering maximally five days after the earliest flowering reference cultivar 'Kišiněvskaja'. All four markers were integrated into a single base extension assay to help breeders with prediction of beginning of flowering for their breeding materials and cultivars.

GBS read coverage analysis identified a Robertsonian chromosome from two Thinopyrum subgenomes in wheat, conferring leaf and stripe rust resistance, drought tolerance, and maintaining yield stability. Agropyron glael (GLAEL), a Thinopyrum intermedium × Th. ponticum hybrid, serves as a valuable genetic resource for wheat improvement. Despite its potential, limited knowledge of its chromosome structure and homoeologous relationships with hexaploid wheat (Triticum aestivum) has restricted the full exploitation of GLAEL's genetic diversity in breeding programs. Here, we present the development of a 44-chromosome wheat/GLAEL addition line (GLA7). Multicolor genomic in situ hybridization identified one chromosome arm from the St subgenome of Th. intermedium, while the other arm remained unclassified. Genotyping-by-sequencing (GBS) read coverage analysis revealed a unique Robertsonian translocation between two distinct Thinopyrum subgenomes, identified as 4StS·1JvsS. The GLA7 line demonstrated strong adult plant resistance to both leaf rust and stripe rust under natural and artificial infection conditions. Automated phenotyping of shoot morphological parameters together with leaf relative water content and yield components showed that the GLA7 line exhibited elevated drought tolerance compared to parental wheat genotypes. Three years of field trials showed that GLA7 exhibits similar agronomic performance and yield components to the wheat parents. This unique addition line holds promise for enhancing wheat's tolerance to multiple stresses through the introduction of new resistance genes, as well as its ability to mitigate the effects of temporary water limitation during flowering, all without negatively impacting wheat performance.