Molekularna identifikacija smeđe alge Cystoseira spp. (Phaeophycae, Fucales) iz Jadranskog mora – preliminarni rezultati

In the attempt to identify an appropriate molecular marker which will enable genetic distinction between different Cystoseira species from the Adriatic Sea, two mitochondrial molecular markers were tested: the mt 23S rDNA and the mt23S-tRNAVal spacer. Two species were studied: Cystoseira spinosa and Cystoseira squarrosa. Sequence analyses showed no variation in the mt 23S rDNA among all individuals analyzed. But the analysis of the mt23S-tRNAVal spacer showed a differentiation between three haplotypes named A, B and C. The most abundant haplotype A was found in equal number in both species, while haplotype B was found only in C. spinosa and haplotype C was found only in C. squarrosa. However, when comparing to sequences available for several selected Mediterranean Cystoseira species, the mt23S-tRNAVal spacer failed to discriminate between species. Although these results indicate a limited use of the mitochondrial mt23S-tRNAVal intergenic spacer for discrimination among Adriatic Cystoseira species, they could also be interpreted as a sign of conspecificity of the investigated species or the reflection of a recent radiation. Further analysis will be necessary to improve molecular identification of these brown algae.U cilju odabira odgovarajućeg molekularnog biljega koji bi omogućio genetičko razlikovanje među različitim vrstama roda Cystoseira u Jadranskom moru, analizirana su DVA molekularna biljega: mitohondrijska 23S rDNA i mitohondrijska razmaknica (mt23S-tRNAVal). U istraživanju su bile korištene dvije vrste Cystoseira spinosa i Cystoseira squarrosa. Sekvenciranje i usporedba tih fragmenata pokazali su da je regija mt23S rDNA viskoko konzervirana, jer u svih analiziranih jedinki te sekvence nisu pokazivale varijablinost. U strukturi mitohondrijske razmaknice (mt23StRNAVal) uočene su dvije mutacije i tri haplotipa; A, B i C. Dok je haplotip B nađen samo u vrste C. spinosa, a haplotip C u vrste C. squarrosa, najdominatniji haplotip A nađen je kod podjednakog broja jedinki obiju vrsta. U usporedbi s nekoliko odabranih vrsta roda Cystoseira, podrijetlom iz drugih dijelova Mediteranskog mora, taj molekularni biljeg mt23S-tRNAVal ipak nije pokazao vrsnu specifičnost. Iako ti preliminarni rezultati pokazuju ograničenu mogućnost mitohondrijske razmaknice (mt23S-tRNAVal) za razlikovanje među jadranskim vrstama roda Cystoseira, oni mogu ukazivati i na konspecifičnost istraživanih vrsta odnosno odražavati njihovu relativno nedavnu divergenciju. Daljnja istraživanja će biti potrebna kako bi se poboljšala molekularna identifikacija vrsta ovih smeđih alga

Molekularna identifikacija smeđe alge Cystoseira spp. (Phaeophycae, Fucales) iz Jadranskog mora – preliminarni rezultati

In the attempt to identify an appropriate molecular marker which will enable genetic distinction between different Cystoseira species from the Adriatic Sea, two mitochondrial molecular markers were tested: the mt 23S rDNA and the mt23S-tRNAVal spacer. Two species were studied: Cystoseira spinosa and Cystoseira squarrosa. Sequence analyses showed no variation in the mt 23S rDNA among all individuals analyzed. But the analysis of the mt23S-tRNAVal spacer showed a differentiation between three haplotypes named A, B and C. The most abundant haplotype A was found in equal number in both species, while haplotype B was found only in C. spinosa and haplotype C was found only in C. squarrosa. However, when comparing to sequences available for several selected Mediterranean Cystoseira species, the mt23S-tRNAVal spacer failed to discriminate between species. Although these results indicate a limited use of the mitochondrial mt23S-tRNAVal intergenic spacer for discrimination among Adriatic Cystoseira species, they could also be interpreted as a sign of conspecificity of the investigated species or the reflection of a recent radiation. Further analysis will be necessary to improve molecular identification of these brown algae.U cilju odabira odgovarajućeg molekularnog biljega koji bi omogućio genetičko razlikovanje među različitim vrstama roda Cystoseira u Jadranskom moru, analizirana su DVA molekularna biljega: mitohondrijska 23S rDNA i mitohondrijska razmaknica (mt23S-tRNAVal). U istraživanju su bile korištene dvije vrste Cystoseira spinosa i Cystoseira squarrosa. Sekvenciranje i usporedba tih fragmenata pokazali su da je regija mt23S rDNA viskoko konzervirana, jer u svih analiziranih jedinki te sekvence nisu pokazivale varijablinost. U strukturi mitohondrijske razmaknice (mt23StRNAVal) uočene su dvije mutacije i tri haplotipa; A, B i C. Dok je haplotip B nađen samo u vrste C. spinosa, a haplotip C u vrste C. squarrosa, najdominatniji haplotip A nađen je kod podjednakog broja jedinki obiju vrsta. U usporedbi s nekoliko odabranih vrsta roda Cystoseira, podrijetlom iz drugih dijelova Mediteranskog mora, taj molekularni biljeg mt23S-tRNAVal ipak nije pokazao vrsnu specifičnost. Iako ti preliminarni rezultati pokazuju ograničenu mogućnost mitohondrijske razmaknice (mt23S-tRNAVal) za razlikovanje među jadranskim vrstama roda Cystoseira, oni mogu ukazivati i na konspecifičnost istraživanih vrsta odnosno odražavati njihovu relativno nedavnu divergenciju. Daljnja istraživanja će biti potrebna kako bi se poboljšala molekularna identifikacija vrsta ovih smeđih alga

Molecular analysis and the effect of the T-DNA insertion in <i>mre11</i> mutant lines.

<p><b>a</b>) Schematic representation of the <i>mre11-4</i> allele with the T-DNA disruption located in the 18^th intron (right border, NPT-1) and the left border (LBc-1) oriented toward 3´ end of the <i>MRE11</i> gene. Vertical arrows indicate the T-DNA insertion sites for <i>mre11-2</i> and <i>mre11-3</i> alleles, previously characterized [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078760#B21" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078760#B35" target="_blank">35</a>]. Green boxes represent exons. <i>MRE11</i> gene specific primers are shown by short horizontal arrows. (<b>b</b>) Reverse transcriptase (RT)-PCR of <i>MRE11</i> transcripts in wild-type and three <i>mre11</i> mutants. The full-length transcripts were not produced in the three <i>mre11</i> mutants. Primers spanning different regions of <i>MRE11</i> transcripts used in the second round of RT-PCR are indicated at the top of each column. Glyceraldehyde-3-phosphate dehydrogenase A (GAPA) was used as control for cDNA amount and quality. <b>c</b>) Schematic representation of the predicted full-length MRE11 protein (wt) and putative truncated MRE11 proteins: <i>mre11-3</i> mutant lacks 461 amino acids, <i>mre11-4</i> lacks 221 amino acids and <i>mre11-2</i> lacks 191 amino acids. Arrows indicate the T-DNA disruption sites of the <i>MRE11</i> gene with respect to the full-length protein. The various putative protein domains are marked according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078760#B8" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078760#B36" target="_blank">36</a>]; the phosphoesterase motifs (I to IV) with red boxes and two DNA binding domains (blue boxes) as well as the regions important for NBS1 and RAD50 interaction. Ideograms are drawn roughly in scale. Scale bars indicate 100 amino acids. <b>d</b>) Sequence analysis of the junction between the T-DNA and <i>MRE11</i> gene obtained via sequencing in the <i>mre11-4</i> mutants. The top line shows the genomic sequence, exon sequence is shown in uppercase letters, intron sequence is shown in lowercase italic letters, the filler DNA nucleotides are shown in small red uppercase letters and the nucleotides derived from the T-DNA insertion are shown in uppercase boldface letters. The bottom lines show the predicted amino acid sequence as a result of the T-DNA insertion. If the truncated intron 18 is not spliced out, hypothetically, 35 amino acids (ARRYRFS CLITFFNSGLLFQTGTTLNPFSGYSFDL) could be derived from the intron, filler DNA and T-DNA and form the C terminus of the predicted protein in the <i>mre11-4</i> line. The predicted STOP codon is indicated by *.</p

Genome instability in mitotic cells from <i>mre11</i> mutants.

<p>Anaphase spreads were prepared from pistils stained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized by epifluorescence microscopy. <b>a</b>) Wild-type figure (upper left) show the phragmoplast, the cytoplasmic structure that forms at the equator of the spindle after the chromosomes have divided during the anaphase of plant mitosis. Genome instability manifested by chromosome fusions and chromosomal breaks is evident in <i>mre11-4</i> and <i>mre11-3</i> cells. Examples of <i>mre11-4</i> anaphase with two bridges and acentric fragment lagging between separating daughter nuclei are shown. Thick fragmented bridge was detected in <i>mre11-3</i> cell. Scale bar indicates 2 μm and serves all micrographs. <b>b</b>) Graphic representation recapitulating the spectrum of cytological abnormalities in mitotic cells from wild-type and <i>mre11</i> flower buds. Chromosomal aberrations in cells are classified in three categories; anaphase bridges, acentric fragments and fragmented bridges. The total number of scored mitotic cells for each genotype is indicated in parentheses. </p

<i>Arabidopsis</i><i>mre11-4</i> and <i>mre11-3</i> mutant alleles confer vegetative growth defects and sterility.

<p><b>a</b>) Morphology of five weeks old <i>mre11</i> mutant plants and their comparison to wild-type plant. The arrows point at regions that are shown at higher magnification in the inserts. Coin for scale = 18 mm. <b>b</b>) Phenotypic appearance of ten-day-old wild-type (wt) and <i>mre11</i> mutant seedlings. wt and <i>mre11-2</i> mutant plants develop true leaves. In contrast, <i>mre11-4</i> and <i>mre11-3</i> (inserts) mutant plants only expand their cotyledons but do not develop true leaves and show reduced root growth. Wild-type and <i>mre11</i> mutant seeds were germinated on MS agar plates. <b>c</b>) Comparison of siliques harvested from mature wild-type and <i>mre11</i> mutants. The siliques from the <i>mr11-4</i> and <i>mre11-3</i> lines produced no seeds. <i>mre11-2</i> siliques were full (normal seed set) and were indistinguishable from wild-type. <i>atm-2</i> mutant plants are partially sterile.</p

Developmental abnormalities of <i>mre11-4</i> and <i>mre11-3</i> mutants were connected with extensive spontaneous cell death.

<p>Necrotic lesions on leaves in wild-type (<b>a</b>,<b>b</b>) and three allelic series of <i>Arabidopsis </i><i>mre11</i> mutants (<b>c</b>-<b>h</b>) were visualized by staining with trypan blue. There was extensive cell death in the leaves of the <i>mre11-4</i> (<b>e</b>,<b>f</b>) and <i>mre11-3</i> (<b>g</b>,<b>h</b>) mutant plants. In contrast, cell death was not observed in the wild-type (<b>a</b>,<b>b</b>) and <i>mre11-2</i> mutant (<b>c</b>,<b>d</b>). Leaf areas marked by red squares are shown at higher magnification below each figure (<b>a1-h1</b>). Scale bar for macroscopic leaf figures indicate 3 mm.</p

<i>Arabidopsis</i><i>mre11-2</i><i>atm-2</i> double mutant plants form empty siliques and are completely sterile.

<p><b>a</b>) Morphology of five weeks old <i>mre11-2 </i><i>atm-2</i> double mutant plant. <b>b</b>) The siliques from the <i>mre11-2 </i><i>atm-2</i> double mutant line produced no seeds, <i>mre11-2</i> siliques had normal seed set and <i>atm-2</i> mutant plants were partially sterile. <b>c</b>) Empty siliques of <i>mre11-2 </i><i>atm-2</i> double mutants - larger magnification. </p

<i>Arabidopsis</i><i>mre11-4</i> mutants exhibit dramatic chromosomal fragmentation and fusion following zygotene/ pachytene.

<p>DAPI stained chromosome spreads from pollen mother cell (PMC) meiocytes are given in (a-d) and (i-p) for wild-type, (e-h) and (q-t) for the <i>mre11-4</i> mutant. The major stages of wild-type meiosis are as follows: leptotene (a), zygotene (b), pachytene (c), diplotene (d), diakinesis (i), metaphase I (j), anaphase I (k), telophase I (l), prophase II (m), metaphase II (n), anaphase II (o), tetrads containing four microspores (p). In <i>mre11-4</i> mutant, after normal leptotene (e) and zygotene (f), all the subsequent stages were severely impaired, hence it was difficult to identify and define meiotic progression precisely. The approximate stages of <i>mre11-4</i> meiosis are as follows: late prophase I (g), post zygo-pachytene fusion/fragmentation (h,q), approximately prophase II (r), approximately anaphase II (s), polyads with variable number of microspores of different sizes (t). All micrographs have the same scale bar = 5 µm.</p