Karyotype characterization of Crotalaria juncea (L.) by chromosome banding and physical mapping of 18S-5.8S-26S and 5S rRNA gene sites

Abstract

The chromosomes of Crotalaria juncea, a legume of agronomic interest with a 2n = 16 karyotype composed of metacentric chromosomes, were analyzed using several cytogenetic techniques. C-banding revealed heterochromatic regions around the centromeres in all chromosomes and adjacent to the secondary constriction on the chromosome 1 short arm. Fluorescent staining with the GC-specific chromomycin A3 (CMA) highlighted these heterochromatic regions and a tiny site on the chromosome 1 long arm while the AT-specific stain 4'-6-diamidino-2-phenylindole (DAPI) induced a reversed pattern. Staining with CMA combined with AT-specific distamycin A (DA) counterstaining quenched the pericentromeric regions of all chromosomes, but enhanced fluorescence was observed at the heterochromatic regions around the secondary constriction and on the long arms of chromosomes 1 and 4. Fluorescence in situ hybridization (FISH) revealed 18S-5.8S-26S rRNA gene sites (45S rDNA) on chromosomes 1 and 4, and one 5S rDNA locus on chromosome 1. All the rDNA sites were co-located with the positive-CMA/DA bands, suggesting they were very rich in GC. Silver staining revealed signals at the main 45S rDNA locus on chromosome 1 and, in some cells, chromosome 4 was labeled. Two small nucleoli were detected in a few interphase cells, suggesting that the minor site on chromosome 4 could be active at some stages of the cell cycle.

Karyotype characterization reveals active 45S rDNA sites located on chromosome termini in Smilax rufescens (Smilacaceae)

“…In situ hybridization was carried out as previously described (Mondin et al, 2007). Briefly, the probe used for 45S rDNA localization was the 9.1-kb maize 45S rDNA repeating unit labeled with biotin-14-dATP by nick translation (Bionick Labeling System, Invitrogen, USA).…”

“…The probe (5 ng/µL) was added to the hybridization mixture and denatured by heating at 95°C for 10 min, cooled and dropped onto the slide preparation that was denatured in a thermocycler at 92°C for 10 min. Hybridization was then carried out at 37°C for 20 h. Post-hybridization steps followed the protocol previously described by Mondin et al (2007). The slides were counterstained with 1 µg/mL DAPI in Vectashield (Vector, USA).…”

Estudios citogenéticos en cuatro taxones de Canna (Cannaceae) de la provincia de Buenos Aires, Argentina

“…Esto sugiere que, a pesar de la similitud en la morfología cariotípica de las especies de Canna, el bandeo DAPI podría detectar la variación interespecífica en la distribución cromosómica de la heterocromátina e identificar pares cromosómicos. Este método permitirá avanzar con la caracterización citogenética de estas especies ya que ha dado buenos resultados en una amplia variedad de géneros tales como Zea L. (González & Poggio 2011, González et al 2013, Fourastié 2015, Citrus L. (Guerra 1993), Crotalaria L. (Mondin et al 2007) y Opuntia Mill. (Realini et al 2014), entre otros.…”

Karyotype characterization of Malpighia emarginata (Malpighiaceae)

“…Our group has used this strategy to determine the karyotype of Stylosanthes (VIEIRA et al, 1993) and Passiflora species (CUCO et al, 2005) as well as for Crotalaria juncea (MONDIN et al, 2007). For this reason, we may speculate here that the variation observed in chromosome 2 is due to a shift of the centromere segment.…”

“…Two kinds of events could explain the non-observation of satellites in metaphase chromosomes: the satellites are in the telomeric region or the expression of nucleolar-organizing regions is not sufficient to result in secondary constriction or satellite formation. In Crotalaria juncea, for instance, Mondin et al (2007) showed that a NOR site was positive but no secondary constriction was visualized at metaphase, the chromosome morphology remaining unmodified.…”