Dorota Siwinska

Evolution and taxonomic split of the model grass Brachypodium distachyon

BACKGROUND AND AIMS Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n = 10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species. METHODS Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated. KEY RESULTS The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species. CONCLUSIONS The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.

Cytogenetic studies of three European species of Centaurea L. (Asteraceae)

Cytogenetic analysis of several populations of Centaurea jacea (2n = 4x = 44), C. oxylepis (2n = 4x = 44) and C. phrygia (2n = 2x = 22) was performed using flow cytometry, differential chromosome staining and FISH. In all species Arabidopsis-type telomeric repeats hybridized only to the terminal part of chromosomes. In C. phrygia three pairs and in C. oxylepis six pairs of chromosomes revealed the hybridization signals of 45S rDNA. Centaurea jacea showed polymorphism in the 45S rDNA loci number, five or six pairs of sites were observed. 5S rDNA loci were located in two pairs of chromosomes in C. phrygia. In C. jacea and C. oxylepis the number and position of 5S rDNA loci were the same: three pairs located interstitially and one terminally. The genome size of the diploid C. phrygia was established as 2.14 pg/2C. The genomes of tetraploid species were nearly two times larger and genome size polymorphism was observed among C. jacea populations.

Molecular cytogenetic analyses of haploid and allopolyploid Pellia species

Abstract The allopolyploid origin of several bryophyte species was reported approximately 20 years ago. Among them the best characterized is Pellia borealis, a hybrid of two cryptic sibling species: Pellia epiphylla-species N and P. epiphylla-species S. Genomes of the allopolyploid liverwort P. borealis and its progenitors were investigated using conventional as well as molecular cytogenetic techniques. The nuclear DNA content and cell cycle phase in the thallus nuclei was established using flow cytometry. Fluorescent differential staining, C-banding and fluorescent in situ hybridization (FISH) with 26S and 5S ribosomal DNA (rDNA) probes revealed new features of the chromosomes in the P. epiphylla–P. borealis complex. Some characteristics found in both the polyploid and haploid karyotypes support earlier suggestions about the allopolyploid origin of P. borealis. The banding pattern observed on P. borealis chromosomes suggests the occurrence of structural changes in the allopolyploid genome. The number and localization of rDNA sequences were established and simultaneous FISH with 26S and 5S rDNA probes showed colocalization of both types of ribosomal RNA (rRNA) genes in Pellia chromosomes. A nuclear DNA estimate showed that the P. borealis nuclear DNA content is about twice that of the largest known nuclear genome previously known in bryophytes.

Molecular and cytogenetic evidence for an allotetraploid origin of Chenopodium quinoa and C. berlandieri (Amaranthaceae)

Most of the cultivated chenopods are polyploids, but their origin and evolutionary history are still poorly understood. Phylogenetic analyses of DNA sequences of four plastid regions, nrITS and nuclear 5S rDNA spacer region (NTS) of two tetraploid chenopods (2n=4x=36), Andean C. quinoa and North American C. berlandieri, and their diploid relatives allowed inferences of their origin. The phylogenetic analyses confirmed allotetraploid origin of both tetraploids involving diploids of two different genomic groups (genomes A and B) and suggested that these two might share very similar parentage. The hypotheses on the origin of the two allopolyploid species were further tested using genomic in situ hybridization (GISH). Several diploid Chenopodium species belonging to the two lineages, genome A and B, suggested by phylogenetic analyses, were tested as putative parental taxa. GISH differentiated two sets of parental chromosomes in both tetraploids and further corroborated their allotetraploid origin. Putative diploid parental taxa have been suggested by GISH for C. quinoa and C. berlandieri. Genome sizes of the analyzed allotetraploids fit nearly perfectly the expected additive values of the putative parental taxa. Directional and uniparental loss of rDNA loci of the maternal A-subgenome was revealed for both C. berlandieri and C. quinoa.

Cytogenetic characterization of the Arabidopsis thaliana natural tetraploid ecotype Warschau stability during in vitro regeneration

The morphological and cytogenetic features of the natural autotetraploid Arabidopsis thaliana ecotype Warschau (Wa-1) were investigated. Most of the Warschau plant organs that were analyzed showed higher size values in comparison with diploid Columbia plants. The tetraploid chromosome number was confirmed by analysis of mitotic metaphase cells and rDNA loci were localized. 35S rDNA loci were present on chromosomes 2 and 4, while 5S rDNA, which is polymorphic among A. thaliana ecotypes, were present on chromosomes 4 and 5. Well-characterized autotetraploid plant material was used for in vitro culture to investigate somaclonal variation. Efficient regeneration through organogenesis was achieved. Most of the plants obtained in vitro exhibited an unchanged ploidy level. Detailed cytogenetic analysis that included chromosome, chromocenters and rDNA signals numbers, revealed the stability of regenerants. Based on these data we recommend the ecotype Warschau as a well-characterized plant material for future investigations on the consequences of polyploidy for the genome.

Chromosomal distribution of rRNA genes in the karyotypes of two dioicous liverwort species from the genus Pellia Raddi

Over one hundred years have passed since the first cytogenetic studies were made on the liverwort genus Pellia Raddi. The karyotype of Pellia is characterised by large chromosomes, a varying heterochromatin content and the presence of sex chromosomes in the dioicous species. Most of the Pellia species are diploids with n = 9, but one of them, Pellia borealis Lorb., has been described as an example of allopolyploidy in liverworts. Although the localisation of rRNA genes, which are essential components of the nuclear genome, remains a challenge in bryophytes, data on the number and chromosomal localisation of 35S and 5S rDNA in all of the Pellia species are now available. Previously, fluorescence in situ hybridisation using rDNA probes was performed on the mitotic chromosomes of 2 monoicous species. The aim of this study was to establish the number and chromosomal distribution of rRNA genes in 2 dioicous diploid species—Pellia endiviifolia (Dicks.) Dumort. and Pellia neesiana (Gottsche) Limpr. The relationships between the species within the genus Pellia can now be discussed in the context of the localisation of the rDNA sites and the range in the number of rDNA loci among bryophytes can also be verified.