Genomic organization of rDNA loci in natural populations of Medicago truncatula Gaertn.

摘要

Annual species constitute the most numerous component of the genus Medicago. They are native to the Mediterranean basin from which they spread to areas of the world with mediterranean climate. These species, commonly named medics, are extremely heterogeneous in morphology, environmental adaptation and in chromosome number. The greater part are diploid with 2n=16 and 2n=14 and two are polyploid with 2n=30 (Quiros and Bauchan 1988). Because of some interesting characteristics such as rapid growth, large production of pods, viability of the seeds on soil for long periods of time and nitrogen fixation by means of the bacterial activity, medics received an increasing attention in the last decade (Crawford et al. 1989; Russi et al. 1992). At present they are regarded as very promising species for sustainable agricultural systems as well as for environmental uses (Bauchan 1998). Numerous studies have also been carried out to understand their phylogenesis and to explore their germplasm (Mariani and Falistocco 1990; Brunner et al. 1995; Diwan et al. 1997). One of the most studied species is M. truncatula, a diploid (2n=16) self-pollinating plant with a small genome size (0.9–1.1 pg) and which responds very well to regeneration and transformation methods (Bennett and Leitch 1995; Chabaud et al. 1996). Such characteristics make M. truncatula a very suitable species for basic genetic studies, and it has been considered a model species for Leguminosae, in general, and of alfalfa (M. sativa L.) in particular (Barker et al. 1990).The analysis of the genetic diversity and the construction of a linkage map based on recombinant lines have been the principal objectives of the research programmes using this species. At present, more then 200 genetic markers have been mapped over 6 linkage groups (Bonnin et al. 1996).In contrast with the advances in the field of molecular biology, cytogenetic research has remained very limited even though it can offer an extremely valuable approach to the analysis of the genome structure of the species and to the understanding of intraspecific variations.In recent years molecular cytogenetic techniques have opened new possibilities for chromosome study, particularly in those species that, like medics, have small and similar chromosomes, thus yielding scarce information when treated with traditional methods (Maluszynska and Heslop-Harrison 1993; Schmidt et al. 1994).DNA fluorescent in situ hybridization (FISH) used in conjunction with fluorochrome staining has turned out to be a crucial tool for effective progress in the study of animal and plant chromosomes by overcoming limitations due to size and morphology. The FISH technique enables the location of genes or other DNA sequences to be visualized directly on metaphase chromosomes. The positions of the labelled sequences may provide useful information in studies of genome organization, chromosome evolution and cytotaxonomy (Jiang and Gill 1994).The sites of the ribosomal genes (18S-5.8S-25S rDNA and 5S rDNA) constitute reliable landmarks for chromosome identification, so the detection of rDNA sequences by FISH is generally the first step toward the physical mapping of chromosomes (Leitch and Heslop-Harrison 1992). Such genes represent two highly conserved families of repetitive DNA sequences in the eukaryotic genome. The 18S-5.8S-25S genes are present in hundreds of repeated units arranged in tandem arrays at the nucleolar organizer regions (NORs) and sometimes at other sites not associated with the NORs (Lapitan 1992). The 5S rRNA have a similar organization, even though their position along the chromosome is not revealed by any morphological characteristics and their detection is possible only by applying in situ hybridization. In the higher eukaryotes such genes constitute a cluster independent from the 18S-5.8S-25S genes.In the present study we analyzed the distribution of 18S-5.8S-25S and 5S ribosomal genes on chromosomes of M. truncatula by using double targ