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.

Long-read data is a great tool to discover new active transposable elements (TEs). However, no ready-to-use tools were available to gather this information from low coverage ONT datasets. Here, we developed a novel pipeline, nanotei, that allows detection of TE-contained structural variants, including individual TE transpositions. We exploited this pipeline to identify TE insertion in the Arabidopsis thaliana genome. Using nanotei, we identified tens of TE copies, including ones for the well-characterized ONSEN retrotransposon family that were hidden in genome assembly gaps. The results demonstrate that some TEs are inaccessible for analysis with the current A. thaliana (TAIR10.1) genome assembly. We further explored the mobilome of the ddm1 mutant with elevated TE activity. Nanotei captured all TEs previously known to be active in ddm1 and also identified transposition of non-autonomous TEs. Of them, one non-autonomous TE derived from (AT5TE33540) belongs to TR-GAG retrotransposons with a single open reading frame (ORF) encoding the GAG protein. These results provide the first direct evidence that TR-GAGs and other non-autonomous LTR retrotransposons can transpose in the plant genome, albeit in the absence of most of the encoded proteins. In summary, nanotei is a useful tool to detect active TEs and their insertions in plant genomes using low-coverage data from Nanopore genome sequencing.

We exploited the advantages of genomic in situ hybridization (GISH) to monitor the introgression process at the chromosome level using a simple and robust molecular marker in the interspecific breeding of bulb onion (Allium cepa L.) that is resistant to downy mildew. Downy mildew (Peronospora destructor [Berk.] Casp.) is the most destructive fungal disease for bulb onions. With the application of genomic in situ hybridization (GISH) and previously developed DMR1 marker, homozygous introgression lines that are resistant to downy mildew were successfully produced in a rather short breeding time. Considering that the bulb onion is a biennial plant, it took seven years from the F₁ hybrid production to the creation of S₂BC₂ homozygous lines that are resistant to downy mildew. Using GISH, it was shown that three progeny plants of S₂BC₂ possessed an A. roylei homozygous fragment in the distal region of the long arm of chromosomes 3 in an A. cepa genetic background. Previously, it was hypothesized that a lethal gene(s) was linked to the downy mildew resistance gene. With the molecular cytogenetic approach, we physically mapped more precisely the lethal gene(s) using the homozygous introgression lines that differed in the size of the A. roylei fragments on chromosome 3.

Evolutionarily related species often share a common order of genes along homeologous chromosomes. Here we report the collinearity disruption of genes located on homeologous chromosome 4 in Allium species. Ultra-sensitive fluorescence in situ hybridization with tyramide signal amplification (tyr-FISH) allowed the visualization of the alliinase multigene family, chalcon synthase gene and EST markers on Allium cepa and Allium fistulosum chromosomes. In A. cepa, bulb alliinase, root alliinase (ALL1) and chalcon synthase (CHS-B) genes were located in the long arm but EST markers (API18 and ACM082) were located in the short arm. In A. fistulosum, all the visualized genes and markers were located in the short arm. Moreover, root alliinase genes (ALL1 and AOB249) showed contrast patterns in number of loci. We suppose that the altered order of the genes/markers is the result of a large pericentric inversion. To get insight into the evolution of the chromosome rearrangement, we mapped the bulb alliinase gene in phylogenetically close and distant species. In the taxonomic clade including A. fistulosum, A. altaicum, A. oschaninii and A. pskemense and in phylogenetically distant species A. roylei and A. nutans, the bulb alliinase gene was located on the short arm of chromosome 4 while, in A. cepa and A. schoenoprasum, the bulb alliinase gene was located on the long arm of chromosome 4. These results have encouraging implications for the further tracing of inverted regions in meiosis of interspecific hybrids and studding chromosome evolution. Also, this finding may have a practical benefit as closely related species are actively used for improving onion crop stock.

Interspecific crossing is a promising approach for introgression of valuable traits to develop cultivars with improved characteristics. Allium fistulosum L. possesses numerous pest resistances that are lacking in the bulb onion (Allium cepa L.), including resistance to Stemphylium leaf blight (SLB). Advanced generations were produced by selfing and backcrossing to bulb onions of interspecific hybrids between A. cepa and A. fistulosum that showed resistance to SLB. Molecular classification of the cytoplasm established that all generations possessed normal (N) male−fertile cytoplasm of bulb onions. Genomic in situ hybridization (GISH) was used to study the chromosomal composition of the advanced generations and showed that most plants were allotetraploids possessing the complete diploid sets of both parental species. Because artificial doubling of chromosomes of the interspecific hybrids was not used, spontaneous polyploidization likely resulted from restitution gametes or somatic doubling. Recombinant chromosomes between A. cepa and A. fistulosum were identified, revealing that introgression of disease resistances to bulb onion should be possible.

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.