Satellite DNA (satDNA) is a family of tandemly repeated non-coding sequences in eukaryotic genomes involved in shaping genome architecture and regulation of various biological functions. Within a species, all satDNA families collectively form the satellitome. Satellitomes of Allium species has been explored only superficially, largely due to enormous genome sizes, high transposable element content, and a general lack of reference genomic resources. The emergence of reference genome assemblies now makes it possible to conduct a more in-depth study. Here, we applied a comprehensive bioinformatics approach to study the satellitomes of Allium cepa and Allium fistulosum. Using two complementary bioinformatics pipelines along with available reference genome assemblies, we have created the most complete collection of consensus satDNA sequences of A. cepa and A. fistulosum so far, consisting of 83 and 97 consensus sequences, respectively. The in silico analysis of the genomic distribution allowed the identification of 11 novel candidates for cytogenetic marker panels, including chromosome-specific satDNA families. Validation of satDNA using PCR and FISH confirmed the reliability of the created satellitomes. Furthermore, comparative analysis of satDNA genomic organization and abundance provided insights into the evolution of these species satellitomes. These findings provide a foundational resource that will help illuminate the evolutionary dynamics of Allium satellitomes and pave the way for future cytogenetic studies of Allium species.

Pollenkitt, a sticky substance produced by the tapetal cells of pollen grains, plays a crucial role in pollen functionality, yet it remains an understudied component in plant biology. In this study, we investigated the role of pollenkitt in onion (Allium cepa) pollen, focusing on its effects on pollen germination, DNase activity, fatty acid composition, and ultrastructure. Our findings reveal that pollenkitt is essential for successful onion pollen germination and tube growth on the stigma, as its removal significantly inhibited these processes. Additionally, we demonstrated that onion pollenkitt exhibits DNase activity, degrading plasmid DNA in a concentration-dependent manner, with EDTA effectively inhibiting the degradation. Gas chromatography identified 20 fatty acids in pollenkitt, with a predominance of unsaturated fatty acids. Using scanning electron microscopy (SEM), we showed structural differences between pollen grains with and without pollenkitt, and we observed the process of pollenkitt removal from the surface of pollen grains by water washing. These results offer valuable insights for future research aimed at optimizing pollen-mediated gene-editing systems and highlight the importance of considering pollenkitt in such applications.

Well-spread pachytene chromosomes are critical for studying the location of meiotic proteins along individual chromosomes. However, producing good spreads in species with large genomes is challenging due to the tangling of pachytene chromosomes. Existing protocols often fail to achieve proper separation of large chromosomes in spreads. Here, we describe in detail an improved protocol that ensures the effective separation of large pachytene chromosomes and demonstrates its suitability for protein immunodetection. To develop the protocol, pollen mother cells at the middle–late pachytene stage from Allium fistulosum, a species with a large genome and chromosomes, were used. The protocol involved three main steps: fixing anthers in Clark’s solution (ethanol–acetic acid, 3:1), digestion in an enzyme mixture, and gentle squashing in 45% acetic acid. A clear ZYP1 signal on all separated chromosomes was observed. The high quality of well-spread pachytene chromosomes obtained with the modified protocol allowed for the easy extraction of individual chromosomes for more precise detection and analysis of the proteins of interest.

Pollen is becoming an increasingly important subject for molecular researchers in genetic engineering, plant breeding, and environmental monitoring. To broaden the scope of these studies, it is essential to develop accessible methods for scientists who are not specialized in palynology. The article presents a simplified technical procedure for preparing pollen grains for scanning electron microscopy (SEM). The protocol is convenient for any molecular laboratory due to its small set of reagents, ease of execution, low cost, does not require special equipment, and takes only one hour to complete. The high penetrating ability of formaldehyde and the final delicate dehydration using hexamethyldisilazane (HMDS) instead of critical point drying allow for sufficient preservation of the architecture of the aperture, which is considered a gateway for the passage of biomolecules. The method was successfully applied to pollen grains of representatives of dicotyledons (beetroot, petunia, radish, tomato and tobacco) and monocotyledons (lily, onion, corn, rye and wheat). Species studied included insect-pollinated (entomophilous) and wind-pollinated (anemophilous) species. A comparative analysis of the sizes of fresh living pollen grains under a light microscope and those prepared for SEM showed some shrinkage. Quantitative analysis of the degree of pollen grain shrinkage showed that this process depends on the initial shape of dry pollen grains, and the number and structure of apertures. The results support the theoretical model of the folding/unfolding pathways of pollen grains.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F-1 diploid (2n = 2x = 8C + 8F) and F-1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F-1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F-1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F-1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F-1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F-1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F-1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

There is little information on the use of pollen in molecular research, despite the increased interest in genome editing by pollen-mediated transformation. This paper presents an essential toolbox of technical procedures and observations for molecular studies on onion (Allium cepa L.) pollen. PCR is a useful tool as an express method to evaluate editing results before pollination. A direct PCR protocol for pollen suspension has been adapted without needing DNA pre-extraction. We showed that the outer layer of lipids known as pollenkitt is a limiting factor for successful PCR on pollen. A simple pre-washing step of pollen suspension was able to eliminate the pollenkitt and enormously affect the PCR results. Additionally, our pollenkitt study helped us develop a simple and effective pollination method using wetted onion pollen grains. Classical manual pollination usually is conducted by intact pollen without wetting. Most existing methods of the editing system delivery into pollen are carried out in a wet medium with consequent drying before pollination, which adversely affects the viability of pollen. The optimal medium for wet pollination was 12% sucrose water solution. Our method of using wetted pollen grains for pollination might be very beneficial for pollen genetic manipulation.

High-resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for the identification of N-, S- and R-cytoplasms, one forward primer was designed to the identical sequences of both cox1 and orf725 genes, and two reverse primers specific to the polymorphic sequences of cox1 and orf725 genes were used. For the second step, breeding lines with N-cytoplasm were evaluated with primers developed from the orfA501 sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial atp9 gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, was S-cytoplasm; the rarest type of CMS was R-cytoplasm; and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%. The identification of the cytoplasm of a single plant by phenotype takes from 4 to 8 years. The HRM-based system enables quick and easy distinguishing of the four types of onion cytoplasm.

The ability to directly look into genome sequences has opened great opportunities in plant breeding. Yet, the assembly of full-length chromosomes remains one of the most difficult problems in modern genomics. Genetic maps are commonly used in de novo genome assembly and are constructed on the basis of a statistical analysis of the number of recombinations. This may affect the accuracy of the ordering and orientation of scaffolds within the chromosome, especially in the region of recombination suppression. Moreover, it is impossible to assign contigs lacking DNA markers. Here, we report the use of Tyr-FISH to determine the position of the short DNA sequence of markers and non-mapped unique copy sequence on the physical chromosomes of a large-genome onion (Allium cepa L.). In order to minimize potential background masking of the target signal, we improved our earlier developed pipeline for probe design. A total of 23 markers were located on physical chromosomes 2 and 6. The order of markers was corrected by the integration of genetic, pseudochromosome maps and cytogenetic maps. Additionally, the position of the mlh1 gene, which was not on the genetic map, was defined on physical chromosome 2. Tyr-FISH mapping showed that the order of 23.1% (chromosome 2) and 27.3% (chromosome 6) of the tested genes differed between physical chromosomes and pseudochromosomes. The results can be used for the improvement of pseudochromosome 2 and 6 assembly. The present study aims to demonstrate the value of the in situ visualization of DNA sequences in chromosome-scaffold genome assembly.

Большинство живых организмов имеют случайное распределение хиазм вдоль хромосом как у лука репчатого (A. cepa). И только немногие являются исключением! Таким исключением является лук-батун (A. fistulosum), у которого хиазмы строго локализованы в проксимальных областях хромосом. Хиазмы – цитологическое проявление кроссинговера. Диаметрально противоположное распределение хиазм возможно является причиной стерильности BC1 гибридов между A. cepa и A. fistulosum. Most living organisms have a random distribution of chiasmata along the chromosomes, like onions (A. cepa). And only a few are exceptions! Such an exception is the onion (A. fistulosum), in which the chiasmata are strictly localized in the proximal regions of the chromosomes. Chiasma is a cytological manifestation of crossing over. The diametrically opposed distribution of chiasmata may be the reason for the sterility of BC1 hybrids between A. cepa and A. fistulosum.

Цитоплазматическая мужская стерильность (CMS) является оптимальной системой для производства гибридного лука F1, благодаря тому, что она обеспечивает снижение затрат и увеличение скорости процесса получения гибридных семян лука. Использование CMS значительно упрощает получение гибридов F1, что приводит к значительному повышение урожайности. Сегодня почти все коммерческие гибридные сорта лука F1 выведены и поддерживаются с помощью CMS. Явление CMS заключается в частичной или полной неспособности пыльников производить жизнеспособные пыльцевые зерна. CMS вызывается мутантными и химерными генами в митохондриальном геноме. Cytoplasmic male sterility (CMS) is the optimal system for F1 hybrid onion production due to the fact that it provides cost savings and increased speed of the hybrid onion seed production process. The use of CMS greatly simplifies the production of F1 hybrids, resulting in a significant increase in yield. Today, almost all commercial hybrid F1 onion varieties are bred and maintained using CMS. The CMS phenomenon is the partial or complete inability of anthers to produce viable pollen grains. CMS is caused by mutant and chimeric genes in the mitochondrial genome.

Открытие системы геномного редактирования CRISPR/Cas9 создает новые возможности в получении сортов растений с заданными свойствами. Генетическое редактирование имеет огромный потенциал для эффективного создания новых сортов растений с заданными свойствами с минимальными затратами времени. Прямое редактирование пыльцевых зерен без использования ДНК-плазмиды, а лишь только с использованием белка Cas9 представляет из себя привлекательную возможность для создания генетически редактированных организмов (ГРО), которые, возможно, будут восприниматься потребителями охотнее, чем ГМО, поскольку при их создании не используется чужеродная ДНК. Кроме этого, такой подход также является менее ресурсозатратным поскольку позволяет избежать этапа регенерации новых растений из редактированных тканей и сразу получить жизнеспособные отредактированные семена со зрелым зародышем. The discovery of the CRISPR/Cas9 genomic editing system creates new opportunities for obtaining plant varieties with desired properties. Genetic editing has a huge potential for the efficient creation of new plant varieties with desired properties with minimal time. Direct editing of pollen grains without the use of a DNA plasmid, but only using the Cas9 protein, is an attractive opportunity to create genetically edited organisms (GROs), which may be more readily accepted by consumers than GMOs, since they do not use foreign DNA. In addition, this approach is also less resource-intensive, since it allows one to avoid the stage of regeneration of new plants from edited tissues and immediately obtain viable edited seeds with a mature embryo.

Технологии секвенирования и сборки геномов продвинулись далеко вперед за последние 10 лет, что позволяет осуществлять сборку de novo видов растений с крупными геномами таких как лук репчатый. Размер генома лука репчатого составляет 16 Gb с огромным содержанием повторяющихся последовательностей (около 92-95%). При сборке генома de novo обычно используются генетические карты, которые строятся на основе статистического анализа числа рекомбинаций. Это может повлиять на точность упорядочения и ориентации скэффолдов внутри хромосомы, особенно в области подавления рекомбинации. Genome sequencing and assembly technologies have advanced greatly over the past 10 years, allowing de novo assembly of plant species with large genomes, such as the onion. The size of the onion genome is 16 Gb with a huge content of repetitive sequences (about 92-95%). When assembling the genome de novo, genetic maps are usually used, which are built on the basis of a statistical analysis of the number of recombinations. This can affect the accuracy of ordering and orientation of scaffolds within a chromosome, especially in the region of recombination suppression.

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.

The centromere is a unique part of the chromosome combining a conserved function with an extreme variability in its DNA sequence. Most of our knowledge about the functional centromere organization is obtained from species with small and medium genome/chromosome sizes while the progress in plants with big genomes and large chromosomes is lagging behind. Here, we studied the genomic organization of the functional centromere in Allium fistulosum and A. cepa, both species with a large genome (13 Gb and 16 Gb/1C, 2n = 2x = 16) and large-sized chromosomes. Using low-depth DNA sequencing for these two species and previously obtained CENH3 immunoprecipitation data we identified two long (1.2 Kb) and high-copy repeats, AfCen1K and AcCen1K. FISH experiments showed that AfCen1K is located in all centromeres of A. fistulosum chromosomes while no AcCen1K FISH signals were identified on A. cepa chromosomes. Our molecular cytogenetic and bioinformatics survey demonstrated that these repeats are partially similar but differ in chromosomal location, sequence structure and genomic organization. In addition, we could conclude that the repeats are transcribed and their RNAs are not polyadenylated. We also observed that these repeats are associated with insertions of retrotransposons and plastidic DNA and the landscape of A. cepa and A. fistulosum centromeric regions possess insertions of plastidic DNA. Finally, we carried out detailed comparative satellitome analysis of A. cepa and A. fistulosum genomes and identified a new chromosome- and A. cepa-specific tandem repeat, TR2CL137, located in the centromeric region. Our results shed light on the Allium centromere organization and provide unique data for future application in Allium genome annotation.

The rapid progress in sequencing technologies and ‘big data’ analysis has opened boundless prospects for study of genome evolution and organization. However, we still have limited number of techniques allowing making a bridge between linear DNA sequence and genome structure. Molecular cytogenetics provides a toolbox of methods for DNA sequence visualization on nucleus or chromosomes bridging the gap between In Silico genome research and In Vivo genome organization. Molecular cytogenetic provides useful information for fundamental study of plant chromosome evolution and has practical application in breeding of new varieties.