Haploid and mixoploid cucumber (Cucumis sativus L.) protoplasts – isolation and fusion

This paper reports on the isolation of haploid and mixoploid protoplasts in the genus Cucumis. The cucumber mixoploid plants (C. sativus L., 2x/4x; 2x = 14) were obtained after oryzalin treatments and the mesophyll protoplasts (2x/4x/8x) were isolated and cultivated by well known in vitro methods. The influence of oryzalin pretreatment on the average viability and density of  protoplasts was tested. The average viability as well as the density is signifi cantly influenced by the oryzalin concentration, whereas the time-span of the treatment doesn’t have significant impact on the density and the viability. Callus formation was the highest level of regeneration in the experiments described in our study. Furthermore the isolation and cultivation of the cucumber and muskmelon (C. melo L.; 2x = 24) haploid protoplasts from young-stage pollen grains were improved. Subsequently, somatic hybridization between mixoploid cucumber protoplasts and muskmelon mesophyll and callus protoplasts, and kiwano (C. metuliferus E. Meyer ex Naudin; 2x = 24) mesophyll protoplasts, by chemical fusion with polyethylene glycol (PEG) 6000 was performed for the first time. Heterofusants were observed and developed into micro colonies. Additionally, the gametosomatic hybridization between mixoploid cucumber protoplasts and pollen muskmelon protoplasts was performed for the first time. Heterofusants and the first cell division were observed, however, the regeneration stopped in this stage. In conclusion, the different ploidy, especially the mixoploid character of isolated protoplasts, has positive influence on protoplasts isolation and the following fusion as represented by a higher regeneration capacity. In addition, both types of protoplasts, haploid and mixoploid, represent a unique systems for biochemical, molecular and genetic experiments. Especially, the haploid protoplasts could be used during in vitro fertilization

New chromosome counts and genome size estimates for 28 species of Taraxacum sect. Taraxacum

The species-rich and widespread genus Taraxacum F. H. Wiggers, 1780 (Asteraceae subfamily Cichorioideae) is one of the most taxonomically complex plant genera in the world, mainly due to its combination of different sexual and asexual reproduction strategies. Polyploidy is usually confined to apomictic microspecies, varying from 3x to 6x (rarely 10x). In this study, we focused on Taraxacum sect. Taraxacum (= T. sect. Ruderalia; T. officinale group), i.e., the largest group within the genus. We counted chromosome numbers and measured the DNA content for species sampled in Central Europe, mainly in Czechia. The chromosome number of the 28 species (T. aberrans Hagendijk, Soest & Zevenbergen, 1974, T. atroviride Štěpánek & Trávníček, 2008, T. atrox Kirschner & Štěpánek, 1997, T. baeckiiforme Sahlin, 1971, T. chrysophaenum Railonsala, 1957, T. coartatum G.E. Haglund, 1942, T. corynodes G.E. Haglund, 1943, T. crassum H. Øllgaard & Trávníček, 2003, T. deltoidifrons H. Øllgaard, 2003, T. diastematicum Marklund, 1940, T. gesticulans H. Øllgaard, 1978, T. glossodon Sonck & H. Øllgaard, 1999, T. guttigestans H. Øllgaard in Kirschner & Štěpánek, 1992, T. huelphersianum G.E. Haglund, 1935, T. ingens Palmgren, 1910, T. jugiferum H. Øllgaard, 2003, T. laticordatum Marklund, 1938, T. lojoense H. Lindberg, 1944 (= T. debrayi Hagendijk, Soest & Zevenbergen, 1972, T. lippertianum Sahlin, 1979), T. lucidifrons Trávníček, ineditus, T. obtusifrons Marklund, 1938, T. ochrochlorum G.E. Haglund, 1942, T. ohlsenii G.E. Haglund, 1936, T. perdubium Trávníček, ineditus, T. praestabile Railonsala, 1962, T. sepulcrilobum Trávníček, ineditus, T. sertatum Kirschner, H. Øllgaard & Štěpánek, 1997, T. subhuelphersianum M.P. Christiansen, 1971, T. valens Marklund, 1938) is 2n = 3x = 24. The DNA content ranged from 2C = 2.60 pg (T. atrox) to 2C = 2.86 pg (T. perdubium), with an average value of 2C = 2.72 pg. Chromosome numbers are reported for the first time for 26 species (all but T. diastematicum and T. obtusifrons), and genome size estimates for 26 species are now published for the first time

Detection of Hybrids in Willows (<i>Salix</i>, Salicaceae) Using Genome-Wide DArTseq Markers

The genus Salix, comprising some 400–500 species, is important in various alluvial or wet habitats of the northern hemisphere. It is a promising crop for applications such as biomass production, biofuels, or environmental projects. Clear species delimitation is crucial in ecology, biotechnology, and horticulture. DArTseq markers, a genome-wide technique, were tested for species and hybrid identification. A total of 179 willow samples were analysed, including six species of Salix subgen. Salix and four species of Salix subgen. Vetrix, including those used in biomass crop production, representing important European taxa. Identification of species-specific markers, clustering analyses (principal coordinate analysis, neighbor-joining) and Bayesian methods (Structure) unambiguously identified putative hybrids. In addition to demonstrating the high efficiency of DArT-seq markers in identifying willow hybrids, we also opened-up new questions about hybridisation processes and systematics. We detected unidirectional hybridisation between S. alba and S. fragilis, forming backcross hybrids, and we rejected the hypothesis that S. fragilis does not occur naturally in Europe. Further, the isolated position of Salix triandra within the genus was confirmed

The pattern of genetic variability in apomictic clones of Taraxacum officinale indicates the alternation of asexual and sexual histories of apomicts.

Dandelions (genus Taraxacum) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis (clonal reproduction through seeds) in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts

DNA content variation and its significance in the evolution of the genus Micrasterias (Desmidiales, Streptophyta).

It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4-30.7 pg in M. truncata and 32.0-64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae

Descriptive statistics for nine apomictic <i>Taraxacum</i> accessions based on six SSR loci. For taxon abbreviations see Table S1.

<p><i>N</i>, sample size; <i>MAS</i>, mean allele size in number of repetition of repeat motif; <i>NDA</i>, number of different alelles; <i>NG</i>, number of different genotypes; <i>G_e</i>, gene diversity counted for loci.</p

Allelic diversity of six nuclear microsatellite loci for <i>Taraxacum</i>.

<p>Allele size, size range of PCR products in number of nucleotides; <i>K</i>, total number of alleles; <i>G_e</i>, gene diversity; <i>F</i>_IS, Wright’s inbreeding coefficient; <i>F</i>_ST, relative differentiation based on allele identity; <i>R</i>_ST relative differentiation based on allele size.</p>***<p>– significant value, P<0.001.</p>1<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041868#pone.0041868-Vaut2" target="_blank">[39]</a>.</p>2<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041868#pone.0041868-Falque1" target="_blank">[38]</a>.</p

SplitDecomposition network.

<p>Network of 96 apomictic individuals of <i>Taraxacum</i> based on 162 AFLP markers (uncorrected <i>P</i>-distances; goodness of fit 85.6). Bootstrap values >50 are shown above branches. Observed cpDNA haplotypes are also designated. For taxon abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041868#pone.0041868.s001" target="_blank">Table S1</a>.</p

Multilocus estimates of <i>F-</i> and <i>R-</i>statistic for all six microsatellite loci for studied apomictic <i>Taraxacum</i> accessions.

***<p>– significant value, P<0.001.</p