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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.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind COs patterning remain largely unknown. In many species COs predominantly occur in the distal 2/3 of the chromosome arm and suppression of COs is observed in the proximal regions. The exceptions are some species, among which Allium fistulosum has strictly localized COs in the proximal region. The ability to manipulate the COs localization can be useful for onion breeding. We investigated the factors that may contribute to the pattern of COs in two closely related onion species A. cepa and A. fistulosum, which differ significantly in the localization of chiasmata, and their F1 diploid and triploid hybrids in pollen mother cells. We demonstrate a significant shift in the COs localization to the distal and interstitial region in F1 triploid hybrid, which has a complete diploid set of A. fistulosum chromosomes and haploid set of A. cepa chromosomes. This observation points to a possible genetic control of COs distribution. We did not find the differences in the assembly and disassembly of ASY1 and ZYP1 between A. cepa and A. fistulosum while the difference between parental species and their hybrids was observed. In diploid F1 hybrids at pachytene the chromosome pairing delay marked by ASY1 was revealed. Immunolocalization of MLH1, a marker for class I of the interference-dependent COs, and MUS81, a marker for class II of the interference-free COs, and mlh1/mus81 gene expression profiling in different stages of meiosis showed a spatiotemporal asymmetry among A. cepa, A. fistulosum and their diploid and triploid F1 hybrids. Our results support the hypothesis of genetic control of CO distribution as one of the players that affect meiotic recombination and exchange of genomic material.

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 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, is S-cytoplasm, the rarest type of CMS is R-cytoplasm, and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%.

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.

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 (

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.

Molecular cytogenetic techniques are valuable tools to study chromosome structure and genome constitution and to perform gene mapping and visualization of chromosomal recombination. In woody ornamental species, which are often characterized by small genomes, small-sized chromosomes and low mitotic index in the tiny-to-handle roots, cytogenetic studies are hardly exploited. We developed the “SteamDrop” protocol, which enabled us to prepare high quality chromosome slides for several woody genera e.g. Buxus, Hydrangea, Buddleja, Hibiscus and Rosa. Study of chromosome number and chromosome morphology, together with knowledge of genetic relationships between species, allowed us to create more straightforward breeding programs for these genera. We also successfully implemented genomic in situ hybridisation (GISH) in different woody ornamental species to prove the hybrid nature of offspring from interspecific crosses within Buddleja and Hibiscus. In addition, knowledge of the physical position of specific genes on chromosomes might promote more efficient breeding and selection. We optimised FISH and Tyramide-FISH on mitotic and pachytene chromosomes of roses. Several single-copy genes (1.1-3 kb) involved in abiotic and biotic stress resistance mechanisms were physically mapped on Rosa wichurana Crep. chromosomes 1, 4 and 7 and genetically mapped on the genetic linkage groups. By this, an anchoring of genetic and physical maps was achieved. In the future, these results will facilitate to pyramid stress related genes in specific genotypes.

Bread-making quality is a crucial trait for wheat and triticale breeding. Several genes significantly influence these characteristics, including glutenin genes and the wheat bread-making (wbm) gene. World wheat collection screening showed that only a few percent of cultivars carry the valuable wbm variant, providing a useful source for wheat breeding. In contrast, no such analysis has been performed for triticale (wheat (AABB genome) &times, rye (RR) amphidiploid) collections. Despite the importance of the wbm gene, information about its origin and genomic organization is lacking. Here, using modern genomic resources available for wheat and its relatives, as well as PCR screening, we aimed to examine the evolution of the wbm gene and its appearance in the triticale genotype collection. Bioinformatics analysis revealed that the wheat Chinese Spring genome does not have the wbm gene but instead possesses the orthologous gene, called wbm-like located on chromosome 7A. The analysis of upstream and downstream regions revealed the insertion of LINE1 (Long Interspersed Nuclear Elements) retrotransposons and Mutator DNA transposon in close vicinity to wbm-like. Comparative analysis of the wbm-like region in wheat genotypes and closely related species showed low similarity between the wbm locus and other sequences, suggesting that wbm originated via introgression from unknown species. PCR markers were developed to distinguish wbm and wbm-like sequences, and triticale collection was screened resulting in the detection of three genotypes carrying wbm-specific introgression, providing a useful source for triticale breeding programs.

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&minus, 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.

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.

Tyramide FISH was used to locate relatively small genomic amplicons from molecular markers linked to Ms locus onto onion chromosome 2 near the centromere, a region of relatively low recombination. Fluorescence in situ hybridization (FISH) has not been readily exploited for physical mapping of molecular markers in plants due to the technical challenge of visualizing small single-copy probes. Signal amplification using tyramide (tyr) FISH can increase sensitivity up to 100-fold. We used tyr-FISH to physically locate molecular markers tightly linked to the nuclear male-fertility (Ms) restoration locus of onion onto mitotic metaphase, pachytene, and super-stretched pachytene chromosomes. Relatively short genomic amplicons (846-2251 bp) and a cDNA clone (666 bp) were visualized in 9-42 % of observed cells. The markers were assigned to proximal locations close to the centromere on the long arm of chromosome 2, a region of lower recombination, revealing that tightly linked markers may be physically distant from Ms. This result explains why several labs have identified molecular markers tightly linked to the Ms locus after screening relatively few DNA clones or primers and segregating progenies. Although these markers are still useful for marker-aided selection, our results indicate that map-based cloning of Ms will likely be difficult due to reduced recombination near this gene.

Allium roylei was employed for the production of alien chromosome addition lines in A. fistulosum. Interspecific hybridization between A. fistulosum and A. roylei successfully produced F1 hybrids. Chromosome doubling of an F1 hybrid was carried out to produce amphidiploids. After two times backcrossing of the amphidiploids with A. fistulosum, a BC2 generation was obtained with chromosome numbers (2n) ranging from 16 to 23. Alien monosomic addition lines (AMAL, FF + nR, 2n = 17) appeared with the highest frequency. Furthermore, multiple addition lines (MAL, 2n = 18–23) were also observed with lower frequencies. Five AMALs (FF + 1R, +3R, +4R, +5R, and +8R) and ten MALs (2n = 18–23) were characterized using isozyme and DNA markers. The extra chromosomes from A. roylei clearly altered the biochemical characteristics of the MALs. Variations in sugar, cysteine sulfoxide, and flavonoid contents were observed among the MALs in various amounts. Allium fistulosum–A. roylei allotriploids (2n = 24, FFR) showed significantly higher saponin content and antifungal activities of saponin extracts against isolates of Fusarium oxysporum f. sp. cepae in comparison with A. fistulosum. This first report of A. fistulosum–A. roylei addition lines opens the possibility of developing novel A. fistulosum cultivars with enhanced nutritional value and disease resistance.

This chapter presents a molecular cytogenetic progress in the study of the Allium chromosomes and its application in onion breeding. A crucial milestone in modern cytogenetics has been the development of fluorescence in situ hybridization (FISH) technology. Thanks to FISH and numerous other technical improvements it became possible to visualize on chromosome repetitive and single-copy DNA sequences that allowed to construct cytogenetic maps and integrate them with recombination maps. A complete set of alien monosomic addition lines (AMALs) of A. fistulosum with an extra chromosome from A. cepa played an essential role for alignment of Allium genetic maps to physical chromosomes. The recent discovery of unusual Allium telomere and the first achievement in the study of the onion centromere provide insight into the biology of this important functional structure of chromosomes. The molecular cytogenetic analysis of the Allium genome has revealed a great species-specific diversity of the chromosome structure, which once again convinces us in the complexity of the plant genome evolution. For the future, much more molecular cytogenetic markers are needed to assist whole-genome assembly in the ongoing SEQUON—Onion Genome Sequencing project. High-density cytogenetic maps will fill the gaps between genome sequencing data and the sub-chromosomal level.

Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon–intron genomic clones for the extremely large genome of onion.

Tandem repeats are often associated with important chromosomal landmarks, such as centromeres, telomeres, subtelomeric, and other heterochromatic regions, and can be good candidates for molecular cytogenetic markers. Tandem repeats present in many plant species demonstrate dramatic differences in unit length, proportion in the genome, and chromosomal organization. Members of genus Allium with their large genomes represent a challenging task for current genetics. Using the next generation sequencing data, molecular, and cytogenetic methods, we discovered two tandemly organized repeats in the Allium fistulosum genome (2n = 2C = 16), HAT58 and CAT36. Together, these repeats comprise 0.25% of the bunching onion genome with 160,000 copies/1 C of HAT58 and 93,000 copies/1 C of CAT36. Fluorescent in situ hybridization (FISH) and C-banding showed that HAT58 and CAT36 associated with the interstitial and pericentromeric heterochromatin of the A. fistulosum chromosomes 5, 6, 7, and 8. FISH with HAT58 and CAT36 performed on A. cepa (2n = 2C = 16) and A. wakegi (2n = 2C = 16), a natural allodiploid hybrid between A. fistulosum and A. cepa, revealed that these repeats are species specific and produced specific hybridization patterns only on A. fistulosum chromosomes. Thus, the markers can be used in interspecific breeding programs for monitoring of alien genetic material. We applied Non-denaturing FISH that allowed detection of the repeat bearing chromosomes within 3 h. A polymorphism of the HAT58 chromosome location was observed. This finding suggests that the rapid evolution of the HAT58 repeat is still ongoing.

The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding activities are going on supported by molecular studies. However, the cytogenetic studies of rose species are scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping. Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore, in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crépin, 1888), a rose species with several benefits for advanced molecular cytogenetic studies of genus Rosa (Kirov et al. 2015a). It is shown that FISH signals from 5S, 45S and an Arabidopsis-type telomeric repeat are distributed on five (1, 2, 4, 5 and 7) of seven chromosome pairs. In addition, it is demonstrated that the interstitial telomeric repeat sequences (ITR) are located in the centromeric regions of four chromosome pairs. Using low hybridization stringency for ITR visualization, we showed that the number of ITR signals increases four times (1–4 signals). This study is the first to propose a FISH-based R. wichurana karyotype for the reliable identification of chromosomes. The possible origin of R. wichurana ITR loci is discussed.

Background Rosaceae is a family containing many economically important fruit and ornamental species. Although fluorescence in situ hybridization (FISH)-based physical mapping of plant genomes is a valuable tool for map-based cloning, comparative genomics and evolutionary studies, no studies using high resolution physical mapping have been performed in this family. Previously we proved that physical mapping of single-copy genes as small as 1.1 kb is possible on mitotic metaphase chromosomes of Rosa wichurana using Tyramide-FISH. In this study we aimed to further improve the physical map of Rosa wichurana by applying high resolution FISH to pachytene chromosomes. Results Using high resolution Tyramide-FISH and multicolor Tyramide-FISH, 7 genes (1.7–3 kb) were successfully mapped on pachytene chromosomes 4 and 7 of Rosa wichurana. Additionally, by using multicolor Tyramide-FISH three closely located genes were simultaneously visualized on chromosome 7. A detailed map of heterochromatine/euchromatine patterns of chromosome 4 and 7 was developed with indication of the physical position of these 7 genes. Comparison of the gene order between Rosa wichurana and Fragaria vesca revealed a poor collinearity for chromosome 7, but a perfect collinearity for chromosome 4. Conclusions High resolution physical mapping of short probes on pachytene chromosomes of Rosa wichurana was successfully performed for the first time. Application of Tyramide-FISH on pachytene chromosomes allowed the mapping resolution to be increased up to 20 times compared to mitotic metaphase chromosomes. High resolution Tyramide-FISH and multicolor Tyramide-FISH might become useful tools for further physical mapping of single-copy genes and for the integration of physical and genetic maps of Rosa wichurana and other members of the Rosaceae. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0233-9) contains supplementary material, which is available to authorized users.

Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies.

Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR) is highly conservative in the segmental hexaploid Thinopyrum intermedium (JrJvsSt) and its possible diploid progenitors Th. bessarabicum (Jb), Pseudoroegneria spicata (St) and Dasypyrum villosum (V) but the abundance of the repeats varied to a large extent. Fluorescence in situ hybridization (FISH) showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In Th. intermedium, the strongest signal of RT-CR probe was detected on the chromosomes of Jv, intermediate on Jr and faint on Js and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in Th. bessarabicum is ~ 2 times and P. spicata is ~ 1,5 times higher than in genome of D. villosum. The possible burst of Ty3/gypsy centromeric retrotransposon in Th. intermedium during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.