High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa

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

Anchoring linkage groups of the Rosa genetic map to physical chromosomes with tyramide-FISH and EST-SNP markers

In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb-1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria

Towards a FISH-based karyotype of Rosa L. (Rosaceae)

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 specks 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 (Crepin, 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 katyotype for the reliable identification of chromosomes. The possible origin of R wichurana ITR loci is discussed

An easy "SteamDrop" method for high quality plant chromosome preparation

BACKGROUND: The chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood. RESULTS: We studied the dynamics of plant chromosome spreading after dropping cell suspension on slides. We showed that steam stimulates cytoplasm hydrolysis and rapid chromosome spreading and that chromosomes stretch during this chromosome spreading. Based on these observations, we developed a novel method, named “SteamDrop”, for the preparation of well-spread mitotic and pachytene chromosomes and successfully used it for 28 plant species with large and small chromosomes. We applied cell suspensions in ethanol instead of the commonly used ethanol/acetic acid fixative. Mitotic and meiotic chromosomes prepared via “SteamDrop” were used in fluorescent in situ hybridization (FISH) experiments with repetitive and unique DNA probes. Long storage of cell suspensions in ethanol did not impair the quality of chromosome preparations. CONCLUSION: The SteamDrop procedure provides a robust and routine method for high quality plant chromosome preparations. The method can be applied for metaphase as well as pachytene chromosome preparation in wide range of species. The chromosomes prepared by SteamDrop are well suitable for repetitive and unique DNA visualization

Introgression of rol genes from rhizogenic Agrobacterium strains into Escallonia spp.

The introgression of rol-genes of rhizogenic Agrobacterium into the plant genome induces changes in plant phenotype and physiology. However, only limited experience with this technique is available for woody ornamentals. To induce new variation within the Escallonia genus, several species were co-cultivated with rhizogenic Agrobacterium strains. Co-cultivation of three rhizogenic Agrobacterium strains (Arqua1, LMG 63 and MAFF210266) with four Escallonia species (E. illinita, E. myrtoidea, E. rosea, and E. rubra), resulted in hairy roots production with a varying efficiency. Co-cultivation of E. rubra with MAFF210266, and E. myrtoidea with LMG63 did not yield hairy roots, while co-cultivation of E. rubra leaves with LMG63 was most successful for hairy root production (up to 80.6%). In addition, the efficiency of hairy root induction depended on the explant type (leaves or nodal sections). The presence of inserted rol-genes and aux-genes in hairy roots was molecularly confirmed using qPCR. Few shoots regenerated from hairy roots, but regeneration needs to be optimized for efficient implementation of rol-genes introgression in Escallonia breeding. Key Message: This research provides a protocol for the production of hairy roots with rol-genes inserted after co-cultivation of several species of Escallonia with rhizogenic Agrobacterium strains

Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum)

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof

The variable effect of polyploidization on the phenotype in Escallonia

To induce new variation within the Escallonia genus, chromosome doubling was performed in E. rubra, E. rosea, and E. illinita, three important species within this genus of mainly evergreen woody ornamental species. Obtained tetraploids and diploid controls were analyzed for rooting capacity, leaf and flower characteristics, and plant architecture using image analysis and cold tolerance. In the present study, a breeders' collection of 23 accessions was characterized cytogenetically and described morphologically. All analyzed species and cultivars were diploid (2n =2x =24), with exception of E. pendula, a tetraploid. Today, breeding in Escallonia is limited to lucky finds in seedling populations and few efforts in interspecific hybridization. Three selected Escallonia species underwent an in vitro chromosome doubling with both oryzalin and trifluralin applied as either a continuous or shock treatment. The treatments successfully induced polyploids in all three species. Image analysis revealed that tetraploid E. rosea had decreased shoot length (from 3.8 to 1.3 cm), higher circularity and more dense growth habit compared to diploids. No significant changes in cold tolerance were seen. Tetraploid E. illinita did not differ in shoot length, but an increased outgrowth of axillary buds on the main axis led to denser plants. For tetraploid E. rubra, an increase in plant height (from 4.9 to 5.5 cm) was observed together with a large decrease in circularity and density due to a more polar outgrowth of branches on the main axis. E. rubra tetraploids bore larger flowers than diploids and had an increased cold tolerance (from 7.7 to 11.8-C). Leaf width and area of tetraploids increased for both E. illinita and E. rubra, while a decrease was seen in E. rosea genotypes. For all three species, the rooting capacity of the tetraploids did not differ from the diploids. We conclude that the effect of polyploidization on Escallonia was highly variable and species dependent

Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines

Interspecific hybridisation in woody ornamentals

Interspecific hybridisation and polyploidisation offer great opportunities to increase variation within existing cultivars and species. Molecular and cytogenetic tools are available to determine the hybrid status of obtained seedlings. However, these modern breeding techniques are rarely used for woody ornamentals. In this study the possibilities of interspecific hybridisation and ploidy breeding for woody ornamentals are investigated. Also the optimal strategies to analyse the hybrid nature of seedlings are determined. Application of morphological, molecular (AFLP) and cytogenetic (GISH, flow cytometry) techniques is evaluated for the case-study genera Hydrangea, Hibiscus and Buddleja. Parental species within these 3 genera were (cyto)genetically very different. This was confirmed by AFLP, flowcytometry and karyotyping. This information gave indications about the crossability of different species within the 3 genera. When performing interspecific crosses there were no prezygotic barriers observed for the 3 genera. However, interspecific hybridisation was hampered due to postzygotic barriers, such as fruit abortion, albinism and growth aberrations in the seedlings. Therefore, in vitro embryo rescue was used for Hydrangea, Hibiscus and Buddleja. Within the 3 genera different hybrid populations were generated. Morphological studies, AFLP and genome size measurements confirmed the hybrid nature of the seedlings. Genomic in situ hybridisation (GISH), is rarely used in woody ornamentals for the identification of the origin of chromosomes in hybrids resulting from breeding programs. In this study, a GISH protocol was optimised for Buddleja and Hibiscus, characterised by small genomes and chromosomes. GISH was successfully used for B. x weyeriana, F1 and F2 hybrids of B. davidii x B. x weyeriana and for hybrids of H. syriacus x H. paramutabilis. The parental genomes could be distinguished and recombinant chromosomes were detected. Polyploidisation by application of mitotic inhibitors on seed(ling)s, was obtained for diploid B. globosa. By using a mitotic polyploidisation protocol a new hexaploid H. syriacus cultivar ‘DVPazurri’ was created and commercialised. Also the application of unreduced gametes offers opportunities to create new genetic variation in a breeding program. In F1 and F2 hybrids of H. syriacus x H. paramutabilis and in B. globosa and B. lindleyana unreduced gametes were observed. This study demonstrated that the creation of interspecific hybrids, along with the chromosome doubling technology offers opportunities for woody ornamental breeding programs. A good understanding of the phenomena associated with allopolyploids and interspecific hybrids lead to important innovations in the commercial assortment of many ornamental genera. In the future these will allow a more efficient and straightforward breeding and selection

Introgression of yellow flower colour in Buddleja davidii by means of polyploidisation and interspecific hybridisation

To introduce yellow colour in the commercial Buddleja davidii (2n = 4x = 76) assortment, an interspecific breeding programme with B. globosa (2n = 2x = 38) was started. The first step was to perform chromosome doubling in B. globosa. Two of the obtained tetraploid B. globosa plants were subsequently used as male parent in interspecific crosses with the white flowering B. davidii cv. Nanhoensis Alba. In total 182 interspecific crosses were made and 18 F1 hybrids were obtained. Genome size measurements, chromosome counts and genomic in situ hybridisation (GISH) analysis proved the hybrid nature of most of the F1 hybrids. Plant morphology also expressed hybrid characteristics. F1 seedlings inherited the yellowish flower colour from B. globosa. As for many other woody ornamentals, the creation of hybrids through interspecific hybridisation along with polyploidisation offers new opportunities for breeding in Buddleja