Tomasz Książczyk

Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.

Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus

The dynamics of genome modification that occurred from the initial hybridization event to the stabilization of allopolyploid species remains largely unexplored. Here, we studied inheritance and expression of rDNA loci in the initial generations of Brassica napus allotetraploids (2n = 38, AACC) resynthesized from Brassica oleracea (2n = 18, CC) and B. rapa (2n = 20, AA) and compared the patterns to natural forms. Starting already from F1 generation, there was a strong uniparental silencing of B. oleracea genes. The epigenetic reprogramming was accompanied with immediate condensation of C-genome nucleolar organizer region (NOR) and progressive transgeneration hypermethylation of polymerase I promoters, mainly at CG sites. No such changes were observed in the A-genome NORs. Locus loss and gains affecting mainly non-NOR loci after the first allotetraploid meiosis did not influence established functional status of NORs. Collectively, epigenetic and genetic modifications in synthetic lines resemble events that accompanied formation of natural allopolyploid species.

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid Secale cereale × Dasypyrum villosum hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.

Genome-dependent chromosome dynamics in three successive generations of the allotetraploid Festuca pratensis × Lolium perenne hybrid.

We focus on the identification of complete and recombined ribosomal DNA-bearing chromosomes, and the dynamics of chromosomal number and position of ribosomal DNA (rDNA) loci in the F2-F4 generations derived from the F1 hybrid of Festuca pratensis Huds. (2n = 4x = 28) × Lolium perenne L. (2n = 4x = 28). Lolium genomic DNA and rRNA genes were mapped by means of genomic and fluorescence in situ hybridization (GISH and FISH). The results revealed that plants of the three generations share various rDNA loci profiles with chromosome structural changes, possibly as a result of chromosomal inter- and intra-rearrangements. We observed an asymmetrical variation in the number of recombinant arms with and without rDNA loci between parental genomes. The Lolium genome was more affected by rearrangements in arms with rDNA loci, while Festuca was more affected in arms without them. Statistically significant differences between L. perenne and F. pratensis genomes concerned the number of recombined chromosomes without rDNA, and the number of recombined rDNA-bearing chromosomal arms of marked chromosomes, showing a tendency of F. pratensis genome-like chromosomes to be less stable, compared with L. perenne. We postulate a novel genome-dependent range and type of chromosome variation in plants of the F2-F4 generations derived from F. pratensis × L. perenne hybrid.

Chinese cabbage (Brassica rapa ssp. pekinensis) – a valuable source of resistance to clubroot (Plasmodiophora brassicae)

Clubroot, caused by the protozoan parasite Plasmodiophora brassicae Woronin, is one of the most damaging diseases of Brassica napus worldwide. Resistant plant material is valuable for cultivation in all areas of high incidence of the disease and intensive growth of oilseed rape. We have evaluated clubroot resistance, plant morphology and seed quality in 15 lines of an F4 generation and selected six lines of F5 generation of interspecific hybrids obtained from a cross between a male sterile line of B. napus ‘MS8’, selected from resynthesized oilseed rape (B. rapa ssp. chinensis × B. oleracea var. gemmifera) and an ecotype of B. rapa ssp. pekinensis. Clubroot resistance was evaluated using a bioassay with P1-P5 pathotypes of P. brassicae (according to the classification of Somé et al. 1996). The resistance to the pathotype P1 was successfully fixed in the F5 generation, and improved in some lines in respect to the pathotypes P2-P4. The resistance to P1 and the other tested pathotypes was not linked. Characterization of plant material included recent techniques of FISH and BAC-FISH with a special focus on the analysis of ribosomal DNA (rDNA) of selected individuals. Two hybrid lines combined high levels of resistance with appropriate plant morphology, good seed quality traits and a stable chromosome number and arrangement. Recent techniques of ‘chromosome painting’ provided good insight into chromosome organization in the hybrids obtained, and offered opportunities of further improvement of the breeding process.

A Survey of Genetic Variation and Genome Evolution within the Invasive Fallopia Complex

The knotweed taxa Fallopia japonica, F. sachalinensis and their interspecific hybrid F. × bohemica are some of the most aggressive invaders in Europe and North America and they are serious threats to native biodiversity. At the same time, they constitute a unique model system for the creation of hybrids and studies of the initiation of evolutionary processes. In the presented study, we focused on (i) examining genetic diversity in selected populations of three Fallopia taxa in the invaded (Poland) and native ranges (Japan), (ii) establishing genome size and ploidy levels and (iii) identifying ribosomal DNA (rDNA)-bearing chromosomes in all of the taxa from the invaded range. We found that the genetic diversity within particular taxa was generally low regardless of their geographical origin. A higher level of clonality was observed for the Polish populations compared to the Japanese populations. Our study suggests that the co-occurrence of F. sachalinensis together with the other two taxa in the same stand may be the source of the higher genetic variation within the F. × bohemica hybrid. Some shift towards the contribution of F. japonica alleles was also observed for selected F. × bohemica individuals, which indicates the possibility of producing more advanced generations of F. × bohemica hybrids. All of the F. sachalinensis individuals were hexaploid (2n = 6x = 66; 2C = 6.01 pg), while those of F. japonica were mostly octoploid (2n = 8x = 88; 2C = 8.87 pg) and all of the F. × bohemica plants except one were hexaploid (2n = 6x = 66; 2C = 6.46 pg). Within the chromosome complement of F. japonica, F. sachalinensis and F. × bohemica, the physical mapping of the rDNA loci provided markers for 16, 13 and 10 chromosomes, respectively. In F. × bohemica, a loss of some of rDNA loci was observed, which indicates the occurrence of genome changes in the hybrid.

Exploiting repetitive sequences and BAC clones in Festuca pratensis karyotyping

The Festuca genus is thought to be the most numerous genus of the Poaceae family. One of the most agronomically important forage grasses, Festuca pratensis Huds. is treated as a model plant to study the molecular mechanisms associated with tolerance to winter stresses, including frost. However, the precise mapping of the genes governing stress tolerance in this species is difficult as its karyotype remains unrecognized. Only two F. pratensis chromosomes with 35S and 5S rDNA sequences can be easily identified, but its remaining chromosomes have not been distinguished to date. Here, two libraries derived from F. pratensis nuclear DNA with various contents of repetitive DNA sequences were used as sources of molecular probes for fluorescent in situ hybridisation (FISH), a BAC library and a library representing sequences most frequently present in the F. pratensis genome. Using FISH, six groups of DNA sequences were revealed in chromosomes on the basis of their signal position, including dispersed-like sequences, chromosome painting-like sequences, centromeric-like sequences, knob-like sequences, a group without hybridization signals, and single locus-like sequences. The last group was exploited to develop cytogenetic maps of diploid and tetraploid F. pratensis, which are presented here for the first time and provide a remarkable progress in karyotype characterization.

Orthology Guided Transcriptome Assembly of Italian Ryegrass and Meadow Fescue for Single-Nucleotide Polymorphism Discovery

Transcriptomes of F. pratensis and L. multiflorum were sequenced and assembled We present a catalogue of SNPs for ancestry analysis and future breeding of grasses We defined interspecific SNPs to study parental‐genome specific gene expression in hybrids We positioned SNPs on linkage groups for high‐resolution genome constitution analysis

Genome Constitution in Selected and Unselected Plants of F2–F4 Generations Derived from an Allotetraploid Festuca pratensis × Lolium perenne Hybrid

The objective of this work was to assess the genomic constitution and intergeneric recombination in three successive unselected generations, (F2–F4), derived from an intergeneric hybrid between Festuca pratensis Huds. (2n = 4x = 28) and Lolium perenne L. (2n = 4x = 28). Examination based on genomic in situ hybridization analyses of randomly chosen plants in each generation indicated progressive changes in genome balance towards that of Lolium. The dominance of Lolium chromatin over Festuca likely resulted from extensive recombination between chromosomes of the parental genomes, together with substitutions of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from generation to generation. The number of recombinant chromosomes, and recombination breakpoints per genotype, also increased in successive generations, but their number was higher for Festuca than for Lolium. The patterns of genome constitutions and recombination were similar to those we observed in selected generations (F2–F4 breeding populations) developed from the same F1 hybrid plants.

Chromosomal Rearrangements in BC 1 Progeny Obtained from Crosses of Tetraploid F. pratensis × L. perenne Hybrids with Tetraploid L . perenne

Physical mapping of genes responsible for quality traits requires well established cytogenetic maps and chromosome identification. Genomic in situ hybridization (GISH) allows to discriminate parental genomes and to track recombination between them. Fluorescence in situ hybridization (FISH) with 5S and 18S–5.8S–25S (35S) rDNA probes provides chromosomal landmarks, and allows to detect chromosome re-arrangements; a characteristic rDNA position provides useful markers for chromosome identification. The aim of this study was to assess the genomic constitution and chromosome arrangements in BC1 progeny obtained from crosses of tetraploid (2n=4x=28) Festuca pratensis × Lolium perenne hybrids with tetraploid (2n=4x=28) L. perenne. GISH examination in BC1 progeny showed variability in respect to somatic Lolium and Festuca chromosome number, as well as Lolium and Festuca recombinant chromosomes and homoeologous recombination breakpoints. FISH experiments showed various numbers of both rDNA loci (3–5 sites for 5S rDNA and 10–13 sites for 35S rDNA). Lolium chromosome 3 and Festuca chromosomes 2 and 3 were also involved in recombination showing rearrangements.