Ana Christina Brasileiro-Vidal

The relationships among lemons, limes and citron: a chromosomal comparison

Lemons, limes and citron constitute a group of closely related Citrus species, whose species delimitations and taxonomic relationships are unclear. In order to identify karyotypic similarities and species relationships within this group, the CMA+/DAPI– banding pattern and the distribution of the 5S and 45S rDNA sites of 10 accessions of lime, lemon, and citron were investigated. The four cultivars of C. limon analyzed showed the same pattern of CMA+ bands and rDNA sites, suggesting that they originated from a single germplasm, later differentiated by distinct somatic mutations. The lemons C. jambhiri, C. limonia and C. volkameriana displayed karyotypes very similar to each other, but they differed from C. limon by the absence of a single chromosome with one band in each telomere. The limes, C. aurantifolia and C. limettioides, seemed less related to each other and exhibited different heteromorphic chromosome pairs. In C. aurantifolia, the presence of a chromosome type unknown in all other Citrus species cytologically known so far supports the assumption that this accession may be derived from a hybrid with a species from the subgenus Papeda or from another genus. Citrus medica was the only homozygous accession of this group and all of its chromosome types were clearly represented in limes and lemons, some of them forming heteromorphic pairs. The analysis of the distribution of rDNA sites allowed a further refinement of the comparison among accessions. The lemons and limes were heterozygous for all rDNA sites, whereas C. medica was entirely homozygous. These data support the hypothesis that C. medica is a true species while the other nine accessions are hybrids.

Mitotic microtubule development and histone H3 phosphorylation in the holocentric chromosomes of Rhynchospora tenuis (Cyperaceae).

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.

Chromosome characterization in Thinopyrum ponticum (Triticeae, Poaceae) using in situ hybridization with different DNA sequences

Thinopyrum ponticum (2n = 10x = 70, JJJJsJs) belongs to the Triticeae tribe, and is currently used as a source of pathogen resistance genes in wheat breeding. In order to characterize its chromosomes, the number and position of 45S and 5S rDNA sites, as well as the distribution of the repetitive DNA sequences pAs1 and pSc119.2, were identified by fluorescent in situ hybridization. The number of nucleoli and NORs was also recorded after silver nitrate staining. Seventeen 45S and twenty 5S rDNA sites were observed on the short arms of 17 chromosomes, the 45S rDNA was always located terminally. On three other chromosomes, only the 5S rDNA site was observed. Silver staining revealed a high number of Ag-NORs (14 to 17) on metaphase chromosomes, whereas on interphase nuclei there was a large variation in number of nucleoli (one to 15), most of them (82.8%) ranging between four and nine. The pAs1 probe hybridized to the terminal region of both arms of all 70 chromosomes. In addition, a disperse labeling was observed throughout the chromosomes, except in centromeric and most pericentromeric regions. When the pSc119.2 sequence was used as a probe, terminal labeling was observed on the short arms of 17 chromosomes and on the long arms of five others. The relative position of 45S and 5S rDNA sites, together with the hybridization pattern of pAs1 and pSc119.2 probes, should allow whole chromosomes or chromosome segments of Th. ponticum to be identified in inbred lines of wheat x Th. ponticum.

Different chromatin fractions of tomato (Solanum lycopersicum L.) and related species.

Conventional chromosome staining has suggested that more than 75% of the tomato chromosomes are constituted by heterochromatin. In order to determine whether more deeply stained proximal regions are classic heterochromatin, the distributions of C-bands and chromomycin A(3) (CMA) bands, and the prophase condensation patterns, were analysed in tomato. In this and most other species of the tomato clade, the 5S and 45S rDNA sites were also localised. In tomato, CMA banding was similar to C-banding. After conventional staining, all species displayed large condensed heteropycnotic regions that did not correspond to C-bands or CMA bands. Analyses of the CMA banded karyotypes revealed a low heterochromatin content. Around 12-17% of the chromatin of tomato was CMA(+) and 1/4 to 1/5 of this heterochromatin corresponded to 45S rDNA. In other species, the CMA(+) heterochromatin showed extensive variation (8-35%), but was never near the values found in the literature for tomato. These data suggest the existence of three principal fractions of chromatin in tomato and related species: the late condensed euchromatin corresponding to the terminal regions of the chromosomes, the precocious condensed euchromatin that occupies the major part of the chromosomes and the constitutive heterochromatin that represents those regions revealed by C-bands.

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.

Heterochromatin and rDNA 5S and 45S sites as reliable cytogenetic markers for castor bean (Ricinus communis, Euphorbiaceae)

The increasing need for renewable energy resources has led to higher demands for biofuel, a scenario where the castor bean (Ricinus communis L.) seed oil represents a promising source of raw material. Despite that, information regarding the genome organization of R. communis is still scarce, impairing the application of modern biotechnological and breeding procedures. The present work brings the first evaluation of the mitotic chromosomes of this species, including 10 potentially interesting accessions for cultivation in semi-arid environments aiming at the biofuel production. The approach included standard staining, fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with rDNA 5S and 45S, as well as silver impregnation. All accessions were diploid with 2n=2x=20, displaying mainly metacentric chromosomes, with CMA-positive bands (GC-rich) in all pairs of the complement. After silver impregnation, one to 14 nucleoli were observed, while the FISH with rDNA 45S revealed two large sites and a variety of minor dots, and the DNAr 5S hybridized in a single pair. The observed features were discussed and compared with literature data regarding pachytene bivalents.

Molecular cytogenetic characterization of parental genomes in the partial amphidiploid Triticum aestivum x Thinopyrum ponticum

The wheat line PF 839197 and six hybrid derivatives from a cross between PF 839197 and Thinopyrum ponticum were cytologically characterized by fluorescent in situ hybridization (FISH). Probes for the 5S and 45S rDNA genes (pTa794 and pTa71, respectively), a highly repetitive rye sequence (pSc119.2), the synthetic oligonucleotide (AAG)5, and total genomic DNA from Th. ponticum and rye were used. In the wheat line, a 1RS.1BL translocation was revealed by the labeling patterns produced with pSc119.2 and (AAG)5, and confirmed by genomic in situ hybridization (GISH) using rye genomic DNA as a probe. Analyses of partial amphiploids confirmed previous results indicating mitotic instability, with a tendency to stabilize at 2n = 42 or 56. GISH with Th. ponticum genomic DNA showed that in one hybrid derivative, with lower chromosome numbers (2n = 42-45), chromosomes were not labeled, whereas in the hybrids with 2n = 48-56 up to 14 chromosomes were labeled. These data suggest that the original chromosome set of these hybrids was 2n = 56, and that chromosomes from both genomes were lost by mitotic instability. FISH using the rDNA probes and GISH with Thinopyrum genomic DNA suggested that cells with 2n = 56 contained an entire wheat genome plus two monoploid chromosome sets of Th. ponticum.

Intra- and interchromosomal rearrangements between cowpea (Vigna unguiculata (L.) Walp.) and common bean (Phaseolus vulgaris L.) revealed by BAC-FISH

Cowpea (Vigna unguiculata) is an annual legume grown in tropical and subtropical regions, which is economically relevant due to high protein content in dried beans, green pods, and leaves. In this work, a comparative cytogenetic study between V. unguiculata and Phaseolus vulgaris (common bean) was conducted using BAC-FISH. Sequences previously mapped in P. vulgaris chromosomes (Pv) were used as probes in V. unguiculata chromosomes (Vu), contributing to the analysis of macrosynteny between both legumes. Thirty-seven clones from P. vulgaris ‘BAT93’ BAC library, corresponding to its 11 linkage groups, were hybridized in situ. Several chromosomal rearrangements were identified, such as translocations (between BACs from Pv1 and Pv8; Pv2 and Pv3; as well as Pv2 and Pv11), duplications (BAC from Pv3), as well as paracentric and pericentric inversions (BACs from Pv3, and Pv4, respectively). Two BACs (from Pv2 and Pv7), which hybridized at terminal regions in almost all P. vulgaris chromosomes, showed single-copy signal in Vu. Additionally, 17 BACs showed no signal in V. unguiculata chromosomes. The present results demonstrate the feasibility of using BAC libraries in comparative chromosomal mapping and karyotype evolution studies between Phaseolus and Vigna species, and revealed several macrosynteny and collinearity breaks among both legumes.

Molecular Markers to Access Genetic Diversity of Castor Bean: Current Status and Prospects for Breeding Purposes

The spurge family (Euphorbiaceae) is one of the most diverse and numerous clades of the angiosperms, including several species of great economic importance as rubber tree (Hevea brasiliensis), cassava (Manihot esculenta), and some oil seed crops, as candlenut (Aleurites moluccana), physic nut (Jatropha curcas) and castor bean (Ricinus communis). Castor bean, the single member of the African genus Ricinus (subfamily Acalyphoideae), presents a wide variation regarding vegetative traits such as leaf and stem colors, number and size of leaf lobes and presence of wax covering the stem (Popova & Moshkin, 1986; Savy-Filho, 2005; Webster, 1994; see Fig. 1). Depending on the environmental conditions, even the vegetative habit may vary, although it is more likely in a shrubby form (Webster, 1994). However, the most conspicuous variability is related to reproductive characters, as color shape and size of seeds, number of flowers per raceme, peduncle length and fruit dehiscence (Figs. 1 and 2) as described by Popova & Moshkin (1986).

Mitotic instability in wheat×Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern

To evaluate the mitotic stability of Triticum aestivum×Thinopyrum ponticum derivatives (BC2F7 and BC2F5 doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring’ and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.