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

Visualization of chromosome territories is a challenging task in plant genomes due to the lack of chromosome-specific probes, especially in species with large genomes. On the other hand, combination of flow sorting, genomic in situ hybridization (GISH), confocal microscopy, and employment of software for 3D modeling enables to visualize and characterize chromosome territories (CT) in interspecific hybrids. Here, we describe the protocol for the analysis of CTs in wheat-rye and wheat-barley hybrids, including amphiploids and introgression forms, where a pair of chromosomes or chromosome arms from one species is introgressed into the genome of another species. In this way, the architecture and dynamics of CTs in various tissues and different stages of cell cycle can be analyzed.

• Klíčová slova
• 3D FISH, Chromosome territory, Confocal microscopy, Imaris, Interphase nucleus, Plant hybrid,
• MeSH
• buněčné jádro genetika   MeSH
• chromozomy rostlin * genetika   MeSH
• genom rostlinný MeSH
• hybridizace in situ MeSH
• zavlečené druhy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even "chaotic" events are dealt with in a proper degree of order.

• Klíčová slova
• 5-ethynyl-2′-deoxyuridine, DNA damage, DNA repair, DNA replication, caffeine, heterochromatin, hydroxyurea, nuclei sorting, premature chromosome condensation, replication stress,
• MeSH
• chromozomy rostlin genetika   MeSH
• fluorescence MeSH
• fyziologický stres * MeSH
• heterochromatin metabolismus   MeSH
• kinetika MeSH
• meristém fyziologie   MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• regenerace fyziologie   MeSH
• replikace DNA * MeSH
• Vicia faba fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• heterochromatin MeSH

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

• MeSH
• buněčný cyklus fyziologie   MeSH
• časové faktory MeSH
• chromozomy rostlin * metabolismus   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• hydroxymočovina MeSH
• metafáze MeSH
• mitotický index MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH
• průtoková cytometrie metody   MeSH
• pufry MeSH
• Saccharum cytologie   genetika   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amiprophos methyl MeSH Prohlížeč
• hydroxymočovina MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH
• pufry MeSH

Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.

• MeSH
• alely MeSH
• buněčný cyklus účinky léků   genetika   MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná metabolismus   MeSH
• fluorescence MeSH
• genom rostlinný * MeSH
• hydroxymočovina farmakologie   MeSH
• karyotyp MeSH
• kinetika MeSH
• kořeny rostlin účinky léků   MeSH
• polyploidie * MeSH
• průtoková cytometrie metody   MeSH
• Saccharum účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• hydroxymočovina MeSH

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

• Klíčová slova
• 3D-FISH, chromatin, genome stability, hybrid, introgression, nucleus, rye, wheat,
• MeSH
• buněčné jadérko MeSH
• centromera MeSH
• chromozomální nestabilita * MeSH
• chromozomy rostlin * MeSH
• hybridizace in situ fluorescenční MeSH
• mitóza MeSH
• pšenice genetika   MeSH
• telomery MeSH
• Publikační typ
• časopisecké články MeSH

Leaf rust, caused by Puccinia triticina, threatens global wheat production due to the constant evolution of virulent pathotypes that defeat commercially deployed all stage-resistance (ASR) genes in modern cultivars. Hence, the deployment of combinations of adult plant resistance (APR) and ASR genes in new wheat cultivars is desirable. Adult plant resistance gene Lr49 was previously mapped on the long arm of chromosome 4B of cultivar VL404 and flanked by microsatellite markers barc163 (8.1 cM) and wmc349 (10.1 cM), neither of which was sufficiently closely linked for efficient marker assisted selection. This study used high-density SNP genotyping and flow sorted chromosome sequencing to fine-map the Lr49 locus as a starting point to develop a diagnostic marker for use in breeding and to clone this gene. Marker sunKASP_21 was mapped 0.4 cM proximal to Lr49, whereas a group of markers including sunKASP_24 were placed 0.6 cM distal to this gene. Testing of the linked markers on 75 Australian and 90 European cultivars with diverse genetic backgrounds showed that sunKASP_21 was most strongly associated with Lr49. Our results also show that the Lr49 genomic region contains structural variation relative to the reference stock Chinese Spring, possibly an inverted genomic duplication, which introduces a new set of challenges for the Lr49 cloning.

• Klíčová slova
• Infinium iSelect 90K SNP array, adult plant resistance, chromosome sorting, leaf rust, marker assisted breeding,
• Publikační typ
• časopisecké články MeSH

The identification of genes of agronomic interest in bread wheat (Triticum aestivum L.) is hampered by its allopolyploid nature (2n = 6x = 42; AABBDD) and its very large genome, which is largely covered by transposable elements. However, owing to this complex structure, aneuploid stocks can be developed in which fragments or entire chromosomes are missing, sometimes resulting in visible phenotypes that help in the cloning of affected genes. In this study, the 2C gametocidal chromosome from Aegilops cylindrica was used to develop a set of 113 deletion lines for chromosome 3D in the reference cultivar Chinese Spring. Eighty-four markers were used to show that the deletions evenly covered chromosome 3D and ranged from 6.5 to 357 Mb. Cytogenetic analyses confirmed that the physical size of the deletions correlated well with the known molecular size deduced from the reference sequence. This new genetic stock will be useful for positional cloning of genes on chromosome 3D, especially for Ph2 affecting homoeologous pairing in bread wheat.

• Klíčová slova
• Ph2, deletion line, gametocidal, homoeologous pairing, wheat,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively. Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC). RESULTS: Repetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes. CONCLUSION: The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.

• MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná analýza   MeSH
• fyzikální mapování chromozomů MeSH
• pšenice genetika   MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekvenční analýza DNA metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH