Characterization and physical mapping of ribosomal RNA gene families in Plantago

Abstract

Background and aims: The organization of rRNA genes in cultivated Plantago ovata Forsk. and several of its wild allies was analysed to gain insight into the phylogenetic relationships of these species in the genus which includes some 200 species.

Methods: Specific primers were designed to amplify the internal transcribed spacer (ITS1 and ITS2) regions from seven Plantago species and the resulting fragments were cloned and sequenced. Similarly, using specific primers, the 5S rRNA genes from these species were amplified and subsequently cloned. Fluorescence in-situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal RNA genes.

Results: The ITS1 region is 19-29 bp longer than the ITS2 in different Plantago species. The 5S rRNA gene-repeating unit varies in length from 289 to 581 bp. Coding regions are highly conserved across species, but the non-transcribed spacers (NTS) do not match any database sequences. The clone from the cultivated species P. ovata was used for physical mapping of these genes by FISH. Four species have one FISH site while three have two FISH sites. In P. lanceolata and P. rhodosperma, the 5S and 45S (18S-5.8S-25S) sites are coupled.

Conclusions: Characterization of 5S and 45S rRNA genes has indicated a possible origin of P. ovata, the only cultivated species of the genus and also the only species with x = 4, from a species belonging to subgenus Psyllium. Based on the studies reported here, P. ovata is closest to P. arenaria, although on the basis of other data the two species have been placed in different subgenera. FISH mapping can be used as an efficient tool to help determine phylogenetic relationships in the genus Plantago and show the interrelationship between P. lanceolata and P. lagopus.

Fig. 1.

Phenetic tree summarizing relationships among Plantago species based on 5S, ITS1 and ITS2 sequences.

Fig. 2.

Physical mapping of 45S (A, C, E, G, I, K and M) and 5S (B, D, F, H, J, L and N) rRNA, sequences, respectively, in seven Plantago species: P. ovata (A and B), P. lanceolata (C and D), P. lagopus (E and F), P. major (G and H), P. rugelii (I and J), P. rhodosperma (K and L) and P. arenaria (M and N).

Fig. 3.

Idiograms of Plantago species showing the position of 18S-5.8S-25S (indicated by a rectangle) and 5S rRNA genes (indicated by a circle and arrow).

Fig. 4.

Southern hybridization pattern of MspI-digested DNA probed with pPov1. The molecular weight marker is indicated in the left margin. Lane 1, P. ovata; lane 2, P. lanceolata; lane 3, P. lagopus; lane 4, P. major; lane 5, P. rugelii; lane 6, P. rhodosperma; lane 7, P. arenaria.

Fig. 5.

Southern hybridization using pTa71 as the probe. Lane 1, P. ovata; lane 2, P. lanceolata; lane 3, P. lagopus; lane 4, P. major; lane 5, P. rugelii; lane 6, P. rhodosperma; lane 7, P. arenaria.