Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred from comparative cytogenetic analysis

Abstract

Background: Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique.

Results: FISH patterns of the repeats in diploid subspecies caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process.

Conclusions: The comparative cytogenetic results provide reliable evidence that diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two subspecies.

Keywords: Chromosome evolution; Diploidization; FISH; M. sativa ssp. caerulea; M. sativa ssp. falcata; Medicago sativa; Repetitive sequences.

Fig. 1

Localization of repeats on Medicago sativa ssp. caerulea PI 464715 somatic chromosomes using a probe E180 (green) in combination with b 18S–26S rDNA, c 5S rDNA, d MsCR-1, e MsCR-2, f MsCR-3, g MsCR-4, h MsCR-5, i MsTR-1, j clone 68, or k clone 87

Fig. 2

Localization of repeats on diploid Medicago sativa ssp. falcata PI 631808 somatic chromosomes using a probe E180 (green) in combination with b 18S–26S rDNA, c 5S rDNA, d MsCR-1, e MsCR-2, f MsCR-3, g MsCR-4, h MsCR-5, i MsTR-1, j clone 68, or k clone 87

Fig. 3

Karyotypes of four Medicago sativa ssp. caerulea accessions and four diploid M. sativa ssp. falcata accessions based on two FISH combinations. a Probed by E180 (green) combined with 18S–26S rDNA (red) and 5S rDNA (red). b Probed by E180 (green) combined with MsTR-1 (red) and MsCR-3 (red)

Fig. 4

Localization of repeats on tetraploid Medicago sativa ssp. falcata XiaNH-072X-824 somatic chromosomes using a probe E180 (green) in combination with b 18S–26S rDNA, c 5S rDNA, d MsCR-1, e MsCR-2, f MsCR-3, g MsCR-4, h MsCR-5, i MsTR-1, j clone 65, k clone 74, l clone 68, and m clone 87

Fig. 5

Karyotypes of six tetraploid Medicago sativa ssp. falcata accessions based on different FISH combinations. a Probed by E180 (green) combined with 18S–26S rDNA (red) and 5S rDNA (red). b Probed by E180 (green) combined with MsTR-1 (red) and MsCR-3 (red)

Fig. 6

Idiogram of FISH-banded chromosomes of (a) Medicago sativa ssp. caerulea, (b) diploid M. sativa ssp. falcata, (c) tetraploid M. sativa ssp. falcata, and (d) M. sativa ssp. sativa. Idiogram of FISH-banded chromosomes of M. sativa ssp. sativa was summarized from four accessions [33]. Chromosomes of tetraploid ssp. sativa are marked with “′” on chromosome numbers. A small black dot next to the FISH signal indicates that the signal is polymorphic across accessions

Fig. 7

a Reconstructing of chromosome-doubling process of diploid ssp. caerulea. Chromosomes of tetraploid ssp. sativa are marked with “′” on chromosome numbers. b Putative chromosome changes during polyploidization process. A small black dot next to the FISH signal indicates that the signal is polymorphic across accessions