Molecular Study of Selected Taxonomically Critical Taxa of the Genus Iris L. from the Broader Alpine-Dinaric Area.

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 ± 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.

Proposal of a New Hybrid Breeding Method Based on Genotyping, Inter-Pollination, Phenotyping and Paternity Testing of Selected Elite F1 Hybrids.

Testing inbred lines for their combining ability is, due to high numbers of line to line testing needed for determination of hybrid performance, the most limiting factor in the F1 hybrid breeding procedure. We propose a novel method of F1 hybrid breeding that enables evaluation of large number of line to line crosses for their hybrid performance. Inbred lines (preferably doubled haploid - DH) are produced from heterozygous populations, genotyped and maintained. A group of lines is inter-pollinated randomly and their progeny examined. To identify elite F1 hybrids, these individual plants are selected by their superior phenotypic characteristics. Finally using paternity testing only of selected hybrids, the origin of paternal lines is revealed. To predict the number of F1 offspring needed in relation to the number of inbred lines being inter-pollinated, a mathematical formula was developed. For instance, using this formula for the inter-pollination of 60 distinct lines, the probability of obtaining all descendants of paternal-parent lines in a maternal-parent row represented at least once is achieved with 420 F1 plants in a row (p = 0.95). In a practical experiment with white cabbage, DH lines were produced using microspore culture; plants were grown to maturity and genotyped at eight polymorphic SSR loci. Two groups of lines (36 and 33 lines per group) were inter-pollinated by two methods, either using cage pollination with bumblebees or using open pollination in isolated field. A total of 9,858 F1 plants were planted and based on their phenotypic characteristics 213 were selected as elite phenotypes. 99 of them were genetically diverse and 5 of them were selected as super elite. Selected plants were analysed by the same SSR markers and the paternal origin of selected F1 plants was determined. Out of 213 selected elite plants 48 were reciprocals thus exhibiting power of selection based on single plant. We demonstrate that this new approach to hybrid development is efficient in white cabbage and we propose breeders to test it in various vegetable and crop species. Moreover, some other aspects of the proposed technique need to be tested and verified both for practical and economic criteria.

The Repetitive DNA Composition in the Natural Pesticide Producer Tanacetum cinerariifolium: Interindividual Variation of Subtelomeric Tandem Repeats.

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), a plant species endemic to the east Adriatic coast, is used worldwide for production of the organic insecticide, pyrethrin. Most studies concerning Dalmatian pyrethrum have focused on its morphological and biochemical traits relevant for breeding. However, little is known about the chromosomal evolution and genome organization of this species. Our study aims are to identify, classify, and characterize repetitive DNA in the T. cinerariifolium genome using clustering analysis of a low coverage genomic dataset. Repetitive DNA represents about 71.63% of the genome. T. cinerariifolium exhibits linked 5S and 35S rDNA configuration (L-type). FISH reveals amplification of interstitial telomeric repeats (ITRs) in T. cinerariifolium. Of the three newly identified satellite DNA families, TcSAT1 and TcSAT2 are located subterminally on most of T. cinerariifolium chromosomes, while TcSAT3 family is located intercalary within the longer arm of two chromosome pairs. FISH reveals high levels of polymorphism of the TcSAT1 and TcSAT2 sites by comparative screening of 28 individuals. TcSAT2 is more variable than TcSAT1 regarding the number and position of FISH signals. Altogether, our data highlights the dynamic nature of DNA sequences associated with subtelomeres in T. cinerariifolium and suggests that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes.

The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 µg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

Chemodiversity of two closely related tetraploid Centaurium species and their hexaploid hybrid: Metabolomic search for high-resolution taxonomic classifiers.

Species within the genus Centaurium readily hybridize and polyploid complexes are often seen in natural populations. We describe phytochemical profiles of newly discovered allohexaploid hybrid, here named Centaurium pannonicum, and its parental tetraploid species C. erythraea and rare C. littorale ssp. compressum. Our aim was to examine chemodiversity of these taxa in the area of Vojvodina (North Serbia) and to perform metabolomics search for chemical classifiers which would provide high resolution discrimination of parental and hybrid individuals. In sum, UHPLC-MS/MS Orbitrap metabolomics fingerprinting revealed seventy compounds in methanol extracts. Despite the lack of qualitative chemical novelty in hybrid plants, UHPLC-qqqMS targeted metabolomics approach, aimed at three secoiridoid compounds and seventeen phenolics, pointed to considerable differences in quantitative composition of these dominant compounds among the plant taxa studied. In addition to the difference in the ploidy levels, the hybrid taxon was well distinguished from both parental species based on metabolite profiles, and, for most individuals, positioned intermediately to the parental taxa in both PCA and hierarchical clustering. After optimizing and comparing several statistical learning methods, it was possible to narrow the number of taxonomic classifiers to five (three xanthones, one secoiridoid glycoside, and one phenolic acid), while increasing the differentiation resolution. The presented metabolomics approach will certainly, along with morphometrics and molecular genetics studies, have high impact on further elucidation of complex relationships among taxa within the genus Centaurium.

DNA labelling of varieties covered by patent protection: a new solution for managing intellectual property rights in the seed industry.

Plant breeders' rights are undergoing dramatic changes due to changes in patent rights in terms of plant variety rights protection. Although differences in the interpretation of ¼breeder's exemption«, termed research exemption in the 1991 UPOV, did exist in the past in some countries, allowing breeders to use protected varieties as parents in the creation of new varieties of plants, current developments brought about by patenting conventionally bred varieties with the European Patent Office (such as EP2140023B1) have opened new challenges. Legal restrictions on germplasm availability are therefore imposed on breeders while, at the same time, no practical information on how to distinguish protected from non-protected varieties is given. We propose here a novel approach that would solve this problem by the insertion of short DNA stretches (labels) into protected plant varieties by genetic transformation. This information will then be available to breeders by a simple and standardized procedure. We propose that such a procedure should consist of using a pair of universal primers that will generate a sequence in a PCR reaction, which can be read and translated into ordinary text by a computer application. To demonstrate the feasibility of such approach, we conducted a case study. Using the Agrobacterium tumefaciens transformation protocol, we inserted a stretch of DNA code into Nicotiana benthamiana. We also developed an on-line application that enables coding of any text message into DNA nucleotide code and, on sequencing, decoding it back into text. In the presented case study, a short command line coding the phrase ¼Hello world« was transformed into a DNA sequence that was inserted in the plant genome. The encoded message was reconstructed from the resulting T1 seedlings with 100 % accuracy. The feasibility and possible other applications of this approach are discussed.

Origins of Allium ampeloprasum horticultural groups and a molecular phylogeny of the section Allium (Allium: Alliaceae).

The subgenus Allium section Allium includes economically important species, such as garlic and leek, as well as other polyploid minor crops. Phylogenetic studies within this section, with a focus on horticultural groups within A. ampeloprasum, were performed on 31 accessions of 17 species using the nuclear ribosomal DNA internal transcribed spacer (ITS) region and the chloroplast trnL-F and trnD-T regions. The results confirmed the monophyly of section Allium. Four main clades were identified on all ITS analyses but the relationships among those and the remaining species studied within section Allium remained unresolved. Trees based on cpDNA recovered two major clades and a topology only partly congruent with that of the ITS tree. Intra-individual polymorphism of the ITS region proved useful in tracking putative parent species of polyploid taxa. The allopolyploid origin of great headed garlic (GHG), A. iranicum and A. polyanthum was confirmed. No signs of hybridization in leek or kurrat were detected but possible introgression events were identified in pearl onion and bulbous leek. Although GHG is often used as a garlic substitute, molecular analysis revealed only a distant relationship with garlic. We also clarified the previous incorrect classification of cultivated forms within A. ampeloprasum, by showing that leek, kurrat, pearl onion, and bulbous leek should be considered separately from GHG.

Chromosome and nuclear DNA study on Luzula - a genus with holokinetic chromosomes.

Chromosomes and nuclear DNA amount were analyzed in leaf tissues of Luzula nivea, Luzula luzuloides, and Luzula multiflora. Intra- and interspecific karyological variability was stated. Chromosome numbers in diploids ranged 2n = 8-24 in L. nivea and L. luzuloides and 2n = 12-84 in hexaploid L. multiflora. Karyological variability resulted mainly from chromosome fission (agmatoploidy) and aneusomaty; chromosome fusion (symploidy) and polyploidy were also involved. Flow cytometric determination of nuclear genome size using propidium iodide staining gave values of 1.584 pg in L. luzuloides, 1.566 pg in L. nivea, and 3.034 pg in L. multiflora. Variability in relative nuclear genome size within species was measured by 4',6-diamidino-2-phenylindole staining. In contrast with previous reports, variability was fairly small and ranged from 1.796 to 1.864 pg in L. luzuloides, from 1.783 to 1.847 pg and from 1.737 to 1.808 pg in two populations (S and F) of L. nivea, respectively, and from 3.125 to 3.271 pg in L. multiflora. An intraplant (interleaf) genome size variation was also observed and its possible causes are discussed.

Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.

Ploidy and morphological characteristics of Solanum tuberosum x Solanum phureja hybrids.

In attempt to induce doubled haploids in potato we studied interspecific hybrids between tetraploid Solanum tuberosum cultivars Igor, Jana, Vesna, Romano, Arinda, Fianna, Donald and Vital and Solanum phureja (clone IVP 48). Four out of eight cultivars produced 21 berries in total and 149 seedlings were obtained. Their ploidy was measured using flow cytometry. Analysis revealed 137 tetraploids, 10 triploids and 2 haploids. One haploid, 6 triploids and most of the tetraploids produced tubers. Nine out of 10 triploids were produced in a cross between cv. Igor and S. phureja. The vigour of the haploid plant was weak and produced characteristic long light yellow tubers. Triploid plants were characterized by a dark violet coloration of the stem, which was the same as the coloration of the S. phureja. Tubers had violet skin colour of various intensities and deep eyes. The majority of the tetraploid plants (135) were phenotypically similar to the S. tuberosum, while two plants had a similar violet stem and tubers as the triploids. Triploids were interspecific hybrids and tetraploids were produced by spontaneous chromosome doubling from S. tuberosum gametes. Two tetraploid plants expressing violet coloration might have been interspecific hybrids formed from 2n S. phureja gametes. Further studies are needed to confirm these assumptions.

Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus.