Molecular evidence for the hybrid origin of Potamogeton ×subrufus Hagstr. (Potamogetonaceae)

Potamogeton ×subrufus Hagstr. was described as a hybrid between P. lucens L. and P. nodosus Poir.; however, the taxon had not been widely accepted and regarded as conspecific with P. ×fluitans Roth, the hybrid between P. lucens and P. natans L. The origin of P. ×subrufus had been obscured till 2010, when, based on morpho-anatomical treatment, it was shown that P. ×subrufus displays several characters consistently different from those of P. ×fluitans. Here we report a successful amplification and sequencing of nuclear ribosomal ITS1 region from a 115 year-old herbarium specimen of P. ×subrufus, collected in locus classicus by J. Baagöe and preserved in the Herbarium of the Institute of Botany, Jagiellonian University (KRA). Based on the additive polymorphism pattern expressed in the ITS1 sequences of P. ×subrufus, we demonstrate that one of the parents of this hybrid was P. nodosus, as was claimed by Hagström

DNA stability contrasts with chromosome variability in Allium fistulosum calli

RAPD analysis was applied to assess the degree of DNA polymorphism in A. fistulosum calli of high chromosomal instability. Nineteen of 24 randomly selected RAPD primers revealed scorable polymorphism between calli and seeds (reference material). Polymorphic band frequency was 55/237 in seeds and 36/233 in calli; variability on the DNA level was thus lower in calli than in seeds (15.4% vs. 23.2% of band positions). UPGMA analysis of Jaccard's coefficients confirmed the genetic similarity of the analyzed cultures. The most distinctive DNA changes in calli involved coincident loss of original bands or the appearance of novel bands. Seven such changes (4 losses, 3 gains) were observed. Our results suggest that changes on the chromosomal level and on the DNA level occurred independently of each other and that different callus lines underwent similar genetic changes during culture, presumably due to strong selection pressure effected by standard in vitro conditions

Male adventitious roots of Rumex thyrsiflorus Fingerh. as a source of genetically stable micropropagated plantlets

Rumex thyrsiflorus Fingerh. is one of the few dioecious plant species that have sex chromosomes. The chromosome constitution of females is 2n = 12A + XX and 2n = 12A + XY1Y2 of males. It is a medicinally important plant species and has also been the object of studies on the structure and function of sex chromosomes and sex ratio. An efficient plant regeneration protocol was developed from karyologically stable male roots that had been derived from a long-term liquid culture. The root segments were grown on MS medium supplemented with the following plant growth regulators: 2.4-D, NAA, kinetin, BAP and TDZ. The highest frequency (81.73 %) of adventitious shoot formation (16.27 shoots/explant) was obtained on MS + 0.5 mg/l TDZ. Regenerated shoots were successfully rooted on ½ MS + 2 % sucrose + 0.5 mg/l IBA and acclimated to in vivo conditions. Histological analysis revealed indirect (via callus) adventitious shoot formation. The cells of the morphogenetic callus were surrounded by a fibrillar structure that was similar to the extracellular matrix. Molecular analysis based on genetic sex markers confirmed that all of the root explants were male. The genetic stability of the regenerated plantlets was confirmed using random amplified polymorphic DNA analysis. This is the first report concerning the micropropagation protocol for R. thyrsiflorus Fingerh. from male roots derived from a long-term liquid culture, which offers a unique opportunity to obtain true-to-type plants of the same sex

Female versus male : Rumex thyrsiflorus Fingerh. under in vitro conditions : does sex influence in vitro morphogenesis?

Rumex thyrsiflorus Fingerh. is a dioecious plant with polymorphic sex chromosomes (XX in females, XY_{1}Y_{2} in males). This species is an interesting plant for study on the structure and function of sex chromosomes, the sex ratio, and is also a valuable source of bioactive constituents. A procedure for the micropropagation of R. thyrsiflorus from hypocotyl explants, which can be useful for analyses of sex-specific morphogenetic reactions under in vitro conditions, was developed. The molecular analysis, based on genetic sex markers, allowed the sex of explants to be determined in order to create male or female in vitro cultures. Regeneration primarily occurred via indirect adventitious shoot formation as well as via somatic embryogenesis, which was confirmed by histological and scanning electron microscope (SEM) analyses. The highest frequency of explants that revealed a morphogenetic response with the highest number of regenerated adventitious shoot buds (average 11.5 shoots per explant) was obtained on a Murashige and Skoog (MS) medium supplemented with 2.27 \mu M thidiazuron (TDZ). Molecular analysis revealed a female-biased sex ratio under these conditions. The relationship between the sex of the explants and their morphogenetic potential was studied. The efficiency of morphogenesis varied between the two sexes and depended on the ratio of auxin and cytokinin in the medium. The germination pattern of male and female seeds under in vitro conditions was also analysed. No differences in germination time between male and female seeds and in terms of male and female seedling length were observed, which indicated that there was no competition between the sexes during seed germination when grown under uniform growth conditions

The first evidence of a host-to-parasite mitochondrial gene transfer in Orobanchaceae

Several parasitic plants are known to have acquired mitochondrial genes via a horizontal transfer from their hosts. However, mitochondrial gene transfer in this direction has not yet been found in the parasite-rich family Orobanchaceae. Based on a phylogenetic analysis of the mitochondrial atp6 gene in selected species of Orobanche s.l., we provide evidence of a host-to-parasite transfer of this gene in O. coerulescens, which is a Eurasiatic species that parasitises Artemisia (Asteraceae). We did not find the original Orobanche atp6 gene in this species, which suggests that it has been replaced by a gene that was acquired from Asteraceae. In addition, our data suggest the occurrence of a second HGT event in the atp6 sequence - from Asteraceae to Phelipanche. Our results support the view that the transfer of genetic material from hosts to parasites influences the mitochondrial genome evolution in the latter

Phylogeny and historical biogeography analysis support Caucasian and Mediterranean centres of origin of key holoparasitic Orobancheae (Orobanchaceae) lineages

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region–Cistanche, Phelypaea, Phelipanche and Orobanche–using nuclear ribosomal (ITS) and plastid (trnL–trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification