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.

• MeSH
• Aegilops * genetics   MeSH
• Chromosomes, Plant genetics   MeSH
• Genetic Linkage MeSH
• Genetic Markers MeSH
• Genome, Plant * MeSH
• Chromosome Mapping MeSH
• Triticum * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Genetic Markers MeSH

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.

• MeSH
• Agropyron genetics   MeSH
• Chromosomes, Plant * genetics   MeSH
• Phenotype MeSH
• Stress, Physiological * genetics   MeSH
• Genotype MeSH
• Genotyping Techniques MeSH
• Plant Diseases * microbiology   genetics   MeSH
• Droughts MeSH
• Disease Resistance * genetics   MeSH
• Triticum * genetics   microbiology   growth & development   MeSH
• Plant Breeding MeSH
• Translocation, Genetic * MeSH
• Publication type
• Journal Article MeSH

UNLABELLED: Tiller number is a key component of wheat plant architecture having a direct impact on grain yield. Because of their viability, biotic resistance, and abiotic stress tolerance, wild relative species are a valuable gene source for increasing wheat genetic diversity, including yield potential. Agropyron glael, a perennial hybrid of Thinopyrum intermedium and Th. ponticum, was created in the 1930s. Recent genome analyses identified five evolutionarily distinct subgenomes (J, Jst, Jvs, Jr, and St), making A. glael an important gene source for transferring useful agronomical traits into wheat. During a bread wheat × A. glael crossing program, a genetically stable translocation line, WT153397, was developed. Sequential in situ hybridizations (McGISH) with J-, St-, and D-genomic DNA probes and pSc119.2, Afa family, pTa71, and (GAA)7 DNA repeats, as well as molecular markers specific for the wheat 6D chromosome, revealed the presence of a 6DS.6Jvs Robertsonian translocation in the genetic line. Field trials in low-input and high-input breeding nurseries over four growing seasons demonstrated the Agropyron chromosome arm's high compensating ability for the missing 6DL, as spike morphology and fertility of WT153397 did not differ significantly from those of wheat parents, Mv9kr1 and 'Mv Karizma.' Moreover, the introgressed 6Jvs chromosome arm significantly increased the number of productive tillers, resulting in a significantly higher grain yield potential compared to the parental wheat cultivars. The translocated chromosome could be highly purified by flow cytometric sorting due to the intense fluorescent labeling of (GAA)7 clusters on the Thinopyrum chromosome arm, providing an opportunity to use chromosome genomics to identify Agropyron gene variant(s) responsible for the tillering capacity. The translocation line WT153397 is an important genetic stock for functional genetic studies of tiller formation and useful breeding material for increasing wheat yield potential. The study also discusses the use of the translocation line in wheat breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-024-01439-y.

• Keywords
• Agropyron glael, FISH, Flow cytometric sorting, GISH, Tillering, Yield potential,
• Publication type
• Journal Article MeSH

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

• Keywords
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• Chromosomes, Plant * MeSH
• Chromosomes * MeSH
• Cytogenetics MeSH
• Karyotyping MeSH
• Flow Cytometry methods   MeSH
• Suspensions MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Suspensions MeSH

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

• Keywords
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publication type
• Journal Article MeSH

Effective utilization of genetic diversity in wild relatives to improve wheat requires recombination between wheat and alien chromosomes. However, this is suppressed by the Pairing homoeologous gene, Ph1, on the long arm of wheat chromosome 5B. A deletion mutant of the Ph1 locus (ph1b) has been used widely to induce homoeologous recombination in wheat × alien hybrids. However, the original ph1b mutation, developed in Chinese Spring (CS) background has poor agronomic performance. Hence, alien introgression lines are first backcrossed with adapted wheat genotypes and after this step, alien chromosome segments are introduced into breeding lines. In this work, the ph1b mutation was transferred from two CSph1b mutants into winter wheat line Mv9kr1. Homozygous genotypes Mv9kr1 ph1b/ph1b exhibited improved plant and spike morphology compared to Chinese Spring. Flow cytometric chromosome analysis confirmed reduced DNA content of the mutant 5B chromosome in both wheat genotype relative to the wild type chromosome. The ph1b mutation in the Mv9kr1 genotype allowed wheat-alien chromosome pairing in meiosis of Mv9kr1ph1b_K × Aegilops biuncialis F1 hybrids, predominantly with the Mb-genome chromosomes of Aegilops relative to those of the Ub genome. High frequency of wheat-Aegilops chromosome interactions resulted in rearranged chromosomes identified in the new Mv9kr1ph1b × Ae. Biuncialis amphiploids, making these lines valuable sources for alien introgressions. The new Mv9kr1ph1b mutant genotype is a unique resource to support alien introgression breeding of hexaploid wheat.

• Keywords
• Aegilops biuncialis, bread wheat, chromosome flow sorting, homoeologous recombination, in situ hybridization, meiotic chromosome pairing, ph1b mutant,
• Publication type
• Journal Article MeSH

Breeding of agricultural crops adapted to climate change and resistant to diseases and pests is hindered by a limited gene pool because of domestication and thousands of years of human selection. One way to increase genetic variation is chromosome-mediated gene transfer from wild relatives by cross hybridization. In the case of wheat (Triticum aestivum), the species of genus Aegilops are a particularly attractive source of new genes and alleles. However, during the evolution of the Aegilops and Triticum genera, diversification of the D-genome lineage resulted in the formation of diploid C, M, and U genomes of Aegilops. The extent of structural genome alterations, which accompanied their evolution and speciation, and the shortage of molecular tools to detect Aegilops chromatin hamper gene transfer into wheat. To investigate the chromosome structure and help develop molecular markers with a known physical position that could improve the efficiency of the selection of desired introgressions, we developed single-gene fluorescence in situ hybridization (FISH) maps for M- and U-genome progenitors, Aegilops comosa and Aegilops umbellulata, respectively. Forty-three ortholog genes were located on 47 loci in Ae. comosa and on 52 loci in Ae. umbellulata using wheat cDNA probes. The results obtained showed that M-genome chromosomes preserved collinearity with those of wheat, excluding 2 and 6M containing an intrachromosomal rearrangement and paracentric inversion of 6ML, respectively. While Ae. umbellulata chromosomes 1, 3, and 5U maintained collinearity with wheat, structural reorganizations in 2, 4, 6, and 7U suggested a similarity with the C genome of Aegilops markgrafii. To develop molecular markers with exact physical positions on chromosomes of Aegilops, the single-gene FISH data were validated in silico using DNA sequence assemblies from flow-sorted M- and U-genome chromosomes. The sequence similarity search of cDNA sequences confirmed 44 out of the 47 single-gene loci in Ae. comosa and 40 of the 52 map positions in Ae. umbellulata. Polymorphic regions, thus, identified enabled the development of molecular markers, which were PCR validated using wheat-Aegilops disomic chromosome addition lines. The single-gene FISH-based approach allowed the development of PCR markers specific for cytogenetically mapped positions on Aegilops chromosomes, substituting as yet unavailable segregating map. The new knowledge and resources will support the efforts for the introgression of Aegilops genes into wheat and their cloning.

• Keywords
• Aegilops comosa, Aegilops umbellulata, chromosome flow sorting and sequencing, chromosome rearrangements, goat grasses, homoeologous relationships, molecular markers, single-gene FISH,
• Publication type
• Journal Article MeSH

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

• MeSH
• In Situ Hybridization, Fluorescence MeSH
• Edible Grain genetics   MeSH
• Metabolic Networks and Pathways genetics   MeSH
• Evolution, Molecular MeSH
• Multigene Family MeSH
• Disease Resistance genetics   MeSH
• Avena genetics   metabolism   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Saponins biosynthesis   chemistry   genetics   MeSH
• Whole Genome Sequencing MeSH
• RNA-Seq MeSH
• Synteny genetics   MeSH
• Nicotiana metabolism   MeSH
• Telomere genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• avenacin A 1 MeSH Browser
• Saponins MeSH

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

• Keywords
• DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
• MeSH
• Cell Cycle MeSH
• Chromosomes, Plant * genetics   MeSH
• Metaphase MeSH
• Flow Cytometry MeSH
• Plants * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

BACKGROUND AND AIMS: Dioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY1Y2 sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes. METHODS: We flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons. KEY RESULTS: We identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes. CONCLUSIONS: The X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.

• Keywords
• Rumex acetosa, genome dynamics, satellites, sex chromosomes, transposable elements,
• MeSH
• Chromosomes, Plant MeSH
• In Situ Hybridization, Fluorescence MeSH
• Evolution, Molecular MeSH
• Sex Chromosomes MeSH
• Retroelements MeSH
• Rumex * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Retroelements MeSH