Sequence Variation of the tRNA ^Leu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

Abstract

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNALeu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNALeu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNALeu (UAA) intron can be of great value when examining cyanobacterial diversity.

Cyanobiont diversity within coralloid roots of selected cycad species

“…A search in GenBank using the intron sequences obtained in the present work identi¢es Nostoc muscorum, Nostoc sp. strain PCC 73102 and/or lichen cyanobiont intron sequences [14,15] as the most similar intron sequences in the database.…”

“…Two primer pairs (outer and inner, respectively), speci¢c for the cyanobacterial tRNA veu (UAA) intron, were used in the PCRs (see [14]). These primers contain restriction sites to facilitate cloning of the obtained DNA fragments.…”

“…The primers were designed by Je¡ Elhai (Department of Biology, University of Richmond, Richmond, VA, USA). PCRs were performed as described previously [14].…”

“…The intron sequences from the cycad cyanobionts show high similarities to the corresponding sequences of the free-living strains Nostoc sp. strain PCC 73102 (originally isolated from the cycad Macrozamia), Nostoc muscorum, and to intron sequences in several lichen cyanobionts [14,15]. However, di¡erences occur in two regions, variable region 1 and 2 respectively (Fig.…”

A small and compact genome in the marine cyanobacterium Prochlorococcus marinus CCMP 1375: lack of an intron in the gene for tRNA(Leu)UAA and a single copy of the rRNA operon

“…The intron in the trnL gene has been used as a phylogenetic marker for cyanobacteria [12,21,22]. The distribution of this intron follows the clustering of cyanobacterial species according to rRNA sequences in phylogenetic trees [12] and it is present in the distantly related chlorophyll b-containing oxyphototroph P. hollandica and in Synechococcus PCC 6301 [12].…”