Phaseolus vulgaris mutants reveal variation in the nuclear genome.

Phaseolus vulgaris L. (common bean) is an essential source of proteins in the human diet worldwide. Bean breeding programs to increase genetic diversity based on induced mutagenesis have a long tradition in Bulgaria. Common bean varieties with high productivity, wide environmental adaptability, good nutritional properties, and improved disease resistance have been successfully developed. In this study, we aimed to investigate selected nuclear genome features, such as the genome size, the number and chromosomal distribution of 5S and 35S rDNA loci by using the fluorescence in situ hybridization (FISH), as well as the level of DNA damage in some local Bulgarian accessions and mutants of P. vulgaris. Flow cytometry analyses revealed no significant differences in genome size between analyzed lines except for one of the analyzed mutants, M19. The value of genome size 2C DNA is about 1.37 pg2C -1 for all lines, whereas it is 1.42 pg2C-1 for M19. The chromosome number remains the same (2n=22) for all analyzed lines. Results of FISH analyses showed that the number of 5S rDNA was stable among accessions and mutant lines (four loci), while the number of 35S rDNA loci was shown as highly polymorphic, varying between ten and sixteen, and displaying differences in the size and location of 35S rDNA loci between analyzed genotypes. The cell cycle profile was different for the analyzed genotypes. The results revealed that wide variation in genome organization and size as well as DNA damage characterizes the analyzed genetic resources of the common bean.

Rubuskaznowskii (Rosaceae), a new bramble species from south-central Poland.

Based on field research in south-central Poland, supplemented with a review of herbarium materials, we identified a stable bramble biotype with a range large enough (190 km distance between the outermost stands) to be described as a new regional agamic species, Rubuskaznowskii sp.nov. It belongs to the series Subthyrsoidei(sect. Corylifolii). Although R.kaznowskii has a unique combination of features, it can be potentially mistaken for R.gothicus. It differs from the latter species in many aspects, including: pruinose primocanes, denser indumentum of the abaxial leaf surface, and more curved prickles on the petiole. R.kaznowskii has mainly been observed on rusty soils, in habitats of mixed coniferous and mixed broadleaf forests, usually in sunny places, along forest margins and roads, in clearings and roadside thickets.

Descending Dysploidy and Bidirectional Changes in Genome Size Accompanied Crepis (Asteraceae) Evolution.

The evolution of the karyotype and genome size was examined in species of Crepis sensu lato. The phylogenetic relationships, inferred from the plastid and nrITS DNA sequences, were used as a framework to infer the patterns of karyotype evolution. Five different base chromosome numbers (x = 3, 4, 5, 6, and 11) were observed. A phylogenetic analysis of the evolution of the chromosome numbers allowed the inference of x = 6 as the ancestral state and the descending dysploidy as the major direction of the chromosome base number evolution. The derived base chromosome numbers (x = 5, 4, and 3) were found to have originated independently and recurrently in the different lineages of the genus. A few independent events of increases in karyotype asymmetry were inferred to have accompanied the karyotype evolution in Crepis. The genome sizes of 33 Crepis species differed seven-fold and the ancestral genome size was reconstructed to be 1C = 3.44 pg. Both decreases and increases in the genome size were inferred to have occurred within and between the lineages. The data suggest that, in addition to dysploidy, the amplification/elimination of various repetitive DNAs was likely involved in the genome and taxa differentiation in the genus.

Ecophysiology of Arabidopsis arenosa, a new hyperaccumulator of Cd and Zn.

Arabidopsis arenosa is a pseudo-metallophyte, closely related to the model hyperaccumulator of Cd and Zn Arabidopsis halleri. A. arenosa occurs naturally in both diploid (2C) and tetraploid (4C) form, in contrast to A. halleri in which only diploid forms were found. Moreover, A. arenosa similarly to A. halleri often occupies heavy metal (HM) contaminated sites. Nevertheless, knowledge about the ecophysiology of this species is very limited. Therefore, we examined fourteen populations of A. arenosa of different ploidy from Central Europe in situ, focusing on photosynthetic efficiency, pigment content and ability to accumulate selected elements. The presented results indicate that several tetraploid populations exhibit the features of Cd and Zn hyperaccumulation. On the one hand, we noted differences in physiological parameters between the studied populations, on the other, harshness of the environment caused similar physiological response such as high HM pollution. All these features suggest that A. arenosa, especially as a new hyperaccumulator of Cd and Zn and autopolyploidyzation model, may be considered a very interesting research object, particularly when investigating the mechanisms of HMs accumulation and tolerance in plants.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Functioning of the Photosynthetic Apparatus in Response to Drought Stress in Oat × Maize Addition Lines.

The oat × maize chromosome addition (OMA) lines, as hybrids between C3 and C4 plants, can potentially help us understand the process of C4 photosynthesis. However, photosynthesis is often affected by adverse environmental conditions, including drought stress. Therefore, to assess the functioning of the photosynthetic apparatus in OMA lines under drought stress, the chlorophyll content and chlorophyll a fluorescence (CF) parameters were investigated. With optimal hydration, most of the tested OMA lines, compared to oat cv. Bingo, showed higher pigment content, and some of them were characterized by increased values of selected CF parameters. Although 14 days of drought caused a decrease of chlorophylls and carotenoids, only slight changes in CF parameters were observed, which can indicate proper photosynthetic efficiency in most of examined OMA lines compared to oat cv. Bingo. The obtained data revealed that expected changes in hybrid functioning depend more on the specific maize chromosome and its interaction with the oat genome rather than the number of retained chromosomes. OMA lines not only constitute a powerful tool for maize genomics but also are a source of valuable variation in plant breeding, and can help us to understand plant susceptibility to drought. Our research confirms more efficient functioning of hybrid photosynthetic apparatus than oat cv. Bingo, therefore contributes to raising new questions in the fields of plant physiology and biochemistry. Due to the fact that the oat genome is not fully sequenced yet, the mechanism of enhanced photosynthetic efficiency in OMA lines requires further research.

Different strategies of Cd tolerance and accumulation in Arabidopsis halleri and Arabidopsis arenosa.

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

In Vitro Tissue Culture in Brachypodium: Applications and Challenges.

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.