Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris

India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of phenotypically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochromes dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culinaris cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes in any Indian crop.

Advances in the cytogenetics of Annonaceae, the case of Annona cherimola L.

Annonaceae represent the largest extant family among the early divergent angiosperms. Despite the long-standing interest in its evolutionary and taxonomic aspects, cytogenetic studies on this family remain extremely few even on economically important species. With this study, we realized a detailed characterization of the chromosomes of Annona cherimola (2n = 14) by a combination of in situ hybridization techniques, fluorochrome banding, and karyomorphological analysis. FISH revealed that 45S and 5S rDNA sites are co-localized in correspondence to the secondary constrictions of the SAT-chromosome pair. Some hypotheses on the organization of the linked 45S and 5S rDNA repeats have been made. FISH with Arabidopsis-type telomeric arrays demonstrated that the A. cherimola telomeres are constituted by TTTAGGG sequences and that they are exclusively localized at the extremities of chromosomes. An insight into the chromosome structure of A. cherimola was obtained by the self-GISH procedure which revealed highly repeated DNA sequences localized in the centromeric regions of all chromosomes. The correspondence of s-GISH signals with DAPI banding suggests that these sequences are the principal component of the centromeric heterochromatin of this species. The karyotype of A. cherimola here described is proposed as the basic reference karyotype for successive investigations in Annonaceae.

Comparative molecular cytogenetic characterization of five wild Vigna species (Fabaceae)

To extend our knowledge on karyotype variation of the genus Vigna Savi, 1824, the chromosomal organization of rRNA genes and fluorochrome banding patterns of five wild Vigna species were studied. Sequential combined PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole) (CPD) staining and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes were used to analyze the karyotypes of V. luteola (Jacquin, 1771) Bentham, 1959, V. vexillata (Linnaeus, 1753) A. Richard, 1845, V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969, V. trilobata (Linnaeus, 1753) Verdcourt, 1968, and V. caracalla (Linnaeus, 1753) Verdcourt,1970. For further phylogenetic analysis, genomic in situ hybridization (GISH) with the genomic DNA of V. umbellata (Thunberg, 1794) Ohwi & H.Ohashi, 1969 onto the chromosomes of five wild Vigna species was also performed. Detailed karyotypes were established for the first time using chromosome measurements, fluorochrome bands, and rDNA-FISH signals. All species had chromosome number 2n = 2x = 22, and symmetrical karyotypes that composed of only metacentric or metacentric and submetacentric chromosomes. CPD staining revealed all 45S rDNA sites in the five species analyzed, (peri)centromeric GC-rich heterochromatin in V. luteola, V. trilobata and V. caracalla, interstitial GC-rich and pericentromeric AT-rich heterochromatin in V. caracalla. rDNA-FISH revealed two 5S loci in V. caracalla and one 5S locus in the other four species; one 45S locus in V. luteola and V. caracalla, two 45S loci in V. vexillata and V. trilobata, and five 45S loci in V. minima. The karyotypes of the studied species could be clearly distinguished by the karyotypic parameters, and the patterns of the fluorochrome bands and the rDNA sites, which revealed high interspecific variation among the five species. The V. umbellata genomic DNA probe produced weak signals in all proximal regions of V. luteola and all (peri)centromeric regions of V. trilobata. The combined data demonstrate that distinct genome differentiation has occurred among the five species during evolution. The phylogenetic relationships between the five wild species and related cultivated species of Vigna are discussed based on our present and previous molecular cytogenetic data.

Cytogenetic studies in three diploid species of Andropogon (Andropogoneae), section Leptopogon

Karyotypes can provide a relevant information about relationships and evolutionary origin among species of the Andropogon genus. This paper presents the karyotype, C+ and DAPI/CMA3 banding and DNA content of three diploid (2n=20) species belonging to section Leptopogon: A. selloanus, A. macrothrix and A. gyrans. Karyotypes of the three diploid species are symmetrical. We propose a karyotype formulae (18m + 2sm) for each of them. The three species show a pair of metacentric chromosomes with a terminal secondary constriction on short arms. Fluorochrome banding revealed different constitutive heterochromatin patterns and CMA3+/DAPI¬ terminal bands related to the nucleolar organizer region in each species. Nuclear DNA content was estimated by flow cytometry ranged from 2.22 to 2.61 pg. FISH technique revealed that these three species have two 45S rDNA loci at the distal ends of the short arms of two metacentric chromosomes. We compare the genomes of the diploids A. selloanus, A. macrothrix and A. gyrans, and the triploid A. ternatus using GISH. These technique allowed us to confirm the hypotheses that the A. selloanus, A. macrothrix and A. gyrans constitute a homogeneous group that share a common S genome that comprises just one of the genomes in the triploid A. ternatus.

Mitosis and Interphase of the Highly Polyploid Palm Voanioalagerardii (2n = 606 ± 3)

The endemic, highly polyploid, monotypic Madagascan palm genus Voanioala (2n ≈ 606) was studied with regard to mitotic stages and interphase. Features of the cell cycle, morphology and sizes of metaphase chromosomes, fluorochrome banding patterns, and silver staining of NORs of such an extremely high polyploid organism are reported for the first time. On a whole, karyokinesis appears to be stable and efficient. A comparison with closely related palm taxa reveals that V. gerardii is 38-ploid, and comparison with the closely related genera Butia, Cocos (coconut) and Jubaea shows that Voanioala has lost ∼35% of its DNA amount subsequent to polyploidization and has suppressed between 74 and 88% of the original nucleolar organizers. About 10 active NORs are present in the nuclei. An auto- or allopolyploid origin of Voanioala is discussed with respect to currently available nuclear gene data. The biogeographic relations to Jubaeopsis, a closely related, monotypic, apparently likewise relict palm genus from eastern mainland South Africa are discussed. From a cytogenetic point of view, a common polyploid ancestor of both genera is most likely, but the available molecular phylogenetic data are not univocal.