Cytological studies of the nucleolus organizing regions in the Medicago complex: sativa–coerulea–falcata

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Abstract
A cytological examination of the nucleolus organizing regions (NORs) of three species from the Medicago sativa complex was conducted to evaluate the structural and functional evolution of the ribosomal RNA (rRNA) loci that encode the 18S, 5.8S, and 26S rRNAs. Mitotic chromosomes in root-tip preparations from tetraploid M. sativa and diploids Medicago coerulea and Medicago falcata were visualized by four methods that provide new data. Fluorescent in situ hybridization using the M. sativa 18S gene as probe localized the structural rDNA to the constricted regions of the satellited chromosomes only. Chromomycin A3 (CMA3) staining and 4′,6-diamidino-2-phenylindole (DAPI) staining identified these chromosomal segments as the most GC-rich regions in the alfalfa karyotype. Medicago falcata exhibited fewer DAPI bands and chromocenters than did M. sativa and M. coerulea. Positive silver nitrate staining showed that all four rDNA regions in M. sativa (located in two chromosome pairs) and both rDNA sites in both diploid species remain transcriptionally active. Counts of nucleoli confirmed that all rDNA regions are independently capable of nucleolus organization. Thus, the number of active NORs in M. sativa is double the number found in M. coerulea or M. falcata. Consequently, if M. sativa originated from sexual hybridization of 2n gametes involving one or both diploid species, no major reorganization or loss of structural or functional rDNA loci has occurred. Key words : alfalfa evolution, CMA3 banding, DAPI banding, fluorescent in situ hybridization, silver nitrate staining.