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.

• Klíčová slova
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• 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
• buněčný cyklus MeSH
• chromozomy rostlin * genetika   MeSH
• metafáze MeSH
• průtoková cytometrie MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Wheat can adapt to most agricultural conditions across temperate regions. This success is the result of phenotypic plasticity conferred by a large and complex genome composed of three homoeologous genomes (A, B, and D). Although drought is a major cause of yield and quality loss in wheat, the adaptive mechanisms and gene networks underlying drought responses in the field remain largely unknown. Here, we addressed this by utilizing an interdisciplinary approach involving field water status phenotyping, sampling, and gene expression analyses. Overall, changes at the transcriptional level were reflected in plant spectral traits amenable to field-level physiological measurements, although changes in photosynthesis-related pathways were found likely to be under more complex post-transcriptional control. Examining homoeologous genes with a 1:1:1 relationship across the A, B, and D genomes (triads), we revealed a complex genomic architecture for drought responses under field conditions, involving gene homoeolog specialization, multiple gene clusters, gene families, miRNAs, and transcription factors coordinating these responses. Our results provide a new focus for genomics-assisted breeding of drought-tolerant wheat cultivars.

• Klíčová slova
• Field drought tolerance, Plant spectral traits, Transcriptomics,
• MeSH
• fyziologický stres * MeSH
• genom rostlinný * MeSH
• lokus kvantitativního znaku MeSH
• období sucha * MeSH
• pšenice genetika   fyziologie   MeSH
• šlechtění rostlin metody   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH

Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of wheat. We performed chromosome-based survey sequencing of the 14 chromosomes, examining repetitive sequences, protein-coding genes, miRNA/target pairs and tRNA genes, as well as syntenic relationships with related grasses. We found considerable differences in the content and distribution of repetitive sequences between the A and B subgenomes. The gene contents of individual chromosomes varied widely, not necessarily correlating with chromosome size. We catalogued candidate agronomically important loci, along with new alleles and flanking sequences that can be used to design exome sequencing. Syntenic relationships and virtual gene orders revealed several small-scale evolutionary rearrangements, in addition to providing evidence for the 4AL-5AL-7BS translocation in wild emmer wheat. Chromosome-based sequence assemblies contained five novel miRNA families, among 59 families putatively encoded in the entire genome which provide insight into the domestication of wheat and an overview of the genome content and organization.

• Klíčová slova
• chromosome sorting, comparative genomics, hexaploid wheat, next-generation sequencing, wild emmer wheat,
• MeSH
• chromozomy rostlin genetika   MeSH
• genetické lokusy genetika   MeSH
• genom rostlinný genetika   MeSH
• konzervovaná sekvence genetika   MeSH
• lipnicovité genetika   MeSH
• mikro RNA genetika   MeSH
• nekódující RNA genetika   MeSH
• polyploidie MeSH
• průtoková cytometrie MeSH
• pšenice genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• rostlinné geny genetika   MeSH
• tetraploidie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• nekódující RNA MeSH