Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

• MeSH
• biotechnologie MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• genom rostlinný * genetika   MeSH
• haplotypy genetika   MeSH
• hybridizace genetická genetika   MeSH
• polyploidie * MeSH
• referenční standardy MeSH
• Saccharum * klasifikace   genetika   MeSH
• šlechtění rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• DNA rostlinná 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.

• Klíčová slova
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• chromozomy rostlin * MeSH
• chromozomy * MeSH
• cytogenetika MeSH
• karyotypizace MeSH
• průtoková cytometrie metody   MeSH
• suspenze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• suspenze 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